Splanchnic and intestinal uptake and formation of estriol and estriol conjugates in the dog in vivo.

A loading dose of 3H-estriol was given to male dogs followed by a constant infusion. The concentrations of total radioactivity, conjugated estriol metabolites, estriol, estriol-o-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-3-sulfate and estriol-3-sulfate, 16alpha-glucosiduronate were determined in plasma from the femoral artery(A), hepatic vein(HV) and superior mesenteric vein (SMV). From these values the splanchnic (100[1-HV/A]) and intestinal (100[1-SMV/A]) extractions were calculated. The mean splanchnic extraction of total radioactivity was positive (23, SE 3, P less than .01), indicating net uptake by the splanchnic area, possibly due to biliary excretion. The mean splanchnic extraction of estriol was 77, SE 1, P less than .01, also indicating net uptake. The splachnic extractions of estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate and estriol-3-sulfate were negative (-15, SE 3, P less than .01; -23, SE 6, P less than .01; -31, SE 8, P less than .01 respectively) indicating net formation of these conjugates for release into the systemic circulation. The mean intestinal extraction of estriol was 12, SE 4, P less than .01, indicating net uptake by the intestine. This net uptake was associated with mean negative intestinal extractions of estriol-3-glucosiduronate (-15, SE 7, P approximately .05), estriol-3-sulfate (-33, SE 10, P less than .01) and estriol-3-sulfate, 16alpha-glucosiduronate (-53, SE 13, P less than .01), indicating net formation of these conjugates by the intestine.     

Estriol metabolism in the baboon: analysis of urinary and biliary metabolites

The metabolism of i.v. estriol was investigated in two intact baboons and four with biliary fistulas. Urine and bile samples were collected periodically and the radioactivity extracted by Amberlite-XAD resin. Metabolites were separated and purified by a combination of DEAE-Sephadex chromatography, celite partition, specific enzyme hydrolysis of the conjugates and identification of the aglycones. The excretion and metabolism of estriol in the animals closely resembled those of the human. Intact animals excreted an average of 50% of the radioactivity in the urine during 12 hours and two animals with biliary drainage excreted an average of 40% in the urine and 49% in the bile. When the steroid was injected into the portal vein an average of 11.5% and 84% were excreted in the urine and bile, respectively.In the urine of intact animals, approximately 65.8% of the radioactivity was in the form of E₃-16G; 14.2% as E₃-3G; 13.4% as E₃-3S and 5.1% as E₃-3S-16G. Over 73% of biliary radioactivity from the peripheral injections was made up of E₃-3S-16G and 3.6% as E₃-16G and 8.3% as 3-sulfate. In the urine,however, 57% of the label was made up of E₃-16G. No radioactive E₃-3G was detected in the bile of any of the animals. Following simultaneous injection of ³H-E₃ peripherally and ¹⁴C-E₃ intraportally, the 3-glucosiduronate excreted in the urine was derived exclusively from the ³H-label. Based on the results obtained, the baboon has been shown to metabolize estriol in the same fashion as the human, with E³-3S-16G as the predominant biliary metabolite and E³-16G as the major urinary metabolite. As in the human, evidence was also found for an enterohepatic circulation of e³ in the baboon, 16-glucuronidation in the kidney, and extrahepatic (enteric?) formation of E³-3G. $\text{In vitro}$ incubation of the baboon liver yielded 94% of the total conjugate as E³-16G without any trace of E³-3G.     

Estrogen metabolism in nonhuman primates I. In vitro biosynthesis of estrogen glucosiduronates in rhesus monkey liver

Estrone glucosiduronate, 17β-estradiol-3-glucoslduronate, 17β-estradiol-17-glucosiduronate and estriol-16α-glucoslduronate have been biosynthesized in substantial yield by incubation of radioactive estrone, 17β-estradiol, estriol and uridlne diphosphoglucosiduronic acid with rhesus monkey liver homogenates. The metabolites were characterized by chromatography on Celite and DEAE-Sephadex, enzyme hydrolysis, derivative formation and crystallization to constant specific activity. The percent conversion to 17β-estradiol-17-glucosiduronate from 17β-estradlol ranged between 56–71%; from estrone, the conversion was 49–54%. Other metabolites formed from estradiol were estrone glucosiduronate(12–21%) and 17β-estradiol-3-glucosiduronate(5–12%). The same metabolites derived from estrone accounted for 18–28% and 10–14% respectively. After estriol incubation, more than 90% of the steroid was converted to estriol-16α-glucosiduronate with no detectable estriol-3-glucosiduronate. This report represents the first time that 17β-estradiol-17-glucosiduronate has been reported as a metabolite in the rhesus monkey and this is the only known species which forms 17β-estradiol-17-glucosiduronate as the predominant metabolite of either estrone or estradiol $\text{in vitro}$.Rhesus monkey liver is similar to the human and baboon in that it metabolizes estriol exclusively to estriol-16-glucosiduronate.     

Estriol in pregnancy. III. Development, comparison and use of specific antisera for rapid radioimmunoassay of unconjugated estriol in pregnancy plasma

Estriol-6-(0-carboxymethyl) oxime (E₃-CMO) and estriol-4-azobenzoic acid (E₃-4-ABA) were linked to bovine serum albumin (BSA). Twelve rabbits were immunized, six with each E₃-BSA conjugate. All six E₃-6-CMO-BSA rabbits, but only one E₃-4-ABA-BSA animal, responded with useful antibody titers. All antisera exhibited good Ring D specificity. E₃-6-CMO-BSA (type 1) antisera cross-reacted up to 220 percent with 6-oxoestriol while the E₃-4-ABA-BSA (type 2) antiserum cross-reacted only 3.8 percent with this steroid. Neither type of antiserum cross-reacted with neutral steroids nor with estriol-16-glucosiduronate and estriol-3-sulfate-16-glucosiduronate, but both cross-reacted with estriol-3-sulfate and estriol-3-glucosiduronate.Both types of antisera could be utilized for a rapid and specific radioimmunoassay (RIA) of unconjugated E₃ in third trimester pregnancy plasma without need for further purification of the plasma extract. Blanks were negligible, sensitivity was sufficient, recovery was virtually complete by using ³H-E₃ as an internal standard, and precision was satisfactory. The measurements of unconjugated plasma E₃ concentrations in ninety apparently normal women between 29 and 40 weeks or gestation obtained by this RIA averaged 7.6, 10.2 and 16.7 ng/ml at 29 to 32, 33 to 36 and 37 to 40 weeks of gestation, respectively.The results obtained in this study indicate that antisera against E₃-6-CMO-BSA, despite their appreciable cross-reaction with 6-oxoestriol, are as useful for a rapid RIA of plasma unconjugated E₃ as antisera against E₃-4-ABA-BSA because very little, if any, 6-oxoestriol is present in late pregnancy plasma. As anti-E₃ titers were much higher and much more readily obtained in response to immunization with E₃-6-CMO-BSA than with E₃-4-ABA-BSA, E₃-6-CMO-BSA appears to be the preferable antigen to develop antisera for a rapid, yet specific, E₃ RIA.     

16-sulfates of estriol in body fluids of human pregnancy at term.

A method was developed for the assay of estriol-16-sulfate (E3-16S) and estriol-3, 16-disulfate (E3-3,16-diS) in maternal serum, cord serum and amniotic fluid at delivery in human pregnancy. Tritiated E3-16S and E3-3,16-diS are added to the fluid being analyzed. The conjugates are separated and purified by sequential chromatography on alumina, Celite and Sephadex LH-20. Each conjugate is hydrolyzed with Glusulase and the released estriol is quantified by radioimmmunoassay. E3-3,16-diS was found in each fluid, most concentrated in the cord serum. Small amounts of E3-16S were found in some amniotic fluids, and this conjugate was virtually absent from the sera. These new estriol conjugates comprise less than 1 percent of total, estriol, apparently too low to be of diagnostic value in human pregnancy.     

The metabolism of estriol in rabbits

Rabbits have been shown to excrete 6, 7-³H-estriol, its conjugates and metabolites preponderantly in the bile during the initial 4 hours following the I.V. injection of the labeled steroid. The amount of radioactivity excreted in the urine was $\text{1}\text{3}$ of that in the bile. Since in intact rabbits most of the injected radioactivity of ³H-estriol is excreted in the urine over a period of days (and very little in the feces), it appears that estriol and its conjugates and metabolites are involved in an efficient enterohepatic. circulation. In the bile, the preponderant metabolite of ³H-estriol was the 3-glucosiduronate. Even though the latter constituted a substantial part of the urinary metabolites, other conjugates and metabolites of estriol were present in considerable amounts. It is possible that the latter have resulted from gastro-intestinal and/or renal metabolism. Incubation of rabbit liver with estriol led to 75% conjugation with glucuronic acid in the 3-position.     

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.     

Urinary excretion of estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate, and estriol-16 alpha-glucosiduronate. Significance of proportionate differences during the menstrual cycle.

The urinary excretion of estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate, and estriol-16 alpha-glucosiduronate in men and throughout the menstrual cycle in women was measured by specific radioimmunoassay. In 9 men the mean +/- SE excretion of these conjugates was 15.9 +/- 1.4, 2.7 +/- 0.3, and 3.2 +/- 0.2 microgram/24 h respectively. In 15 women studied in the midfollicular phase (day 8) of the menstrual cycle, the excretion was 19.4 +/- 1.7, 2.9 +/- 0.2, and 5.4 +/- 1.3 micrograms/24 h. Excretion of each conjugate was significantly (P less than 0.01) elevated in the midluteal phase (day 22) to 41.9 +/- 3.9, 6.3 +/- 0.8, and 12.2 +/- 1.5 micrograms/24 h respectively (n = 14). The mean excretion of estriol-16 alpha-glucosiduronate was greater than that of 17 beta-estradiol-17-glucosiduronate in the luteal phase (P less than 0.05) but not in the follicular phase or in men (P greater than 0.05). The excretion of each of these specific conjugates measured throughout the menstrual cycle in 7 women was characterized by a sharp midcycle peak and a lower, broader luteal phase peak. The ratios of estriol-16 alpha-glucosiduronate to 17 beta-estradiol-17-glucosiduronate, estrone glucosiduronate to 17 beta-estradiol-17-glucosiduronate, and estriol-16 alpha-glucosiduronate to estrone glucosiduronate throughout the menstrual cycle were analyzed. When the mean ratio during the follicular phase was set at 1, a significant increase (P less than 0.01) occurred in the mean luteal phase ratio in each case: 1.00 +/- 0.03 to 1.66 +/- 0.09, 1.00 +/- 0.04 to 1.30 +/- 0.04, and 1.00 +/- 0.03 to 1.24 +/- 0.04 (mean +/- SE) respectively. The marked alteration in the proportions of these urinary estrogen conjugates may be due to altered metabolism of 17 beta-estradiol, but it more likely reflects a change in the pattern of estrogen secretion or production between the two phases of the menstrual cycle.     

Biology and receptor interactions of estriol and estriol derivatives in vitro and in vivo

The biological effects of estriol (E3) have been studied in three estrogen targets, namely, the rat uterus in vivo and in vitro, in primary human endometrial cell cultures and in MCF-7 human breast cancer cells in culture. Studies on the temporal relationships between estrogen receptor binding and biological responses in the uterus using estriol and several more long-acting estriol derivatives, namely, 17α-ethynyl estriol, estriol-3-cyclopentyl ether, and 17α-ethynyl estriol-3-cyclopentyl ether, indicate that estriol is a short-acting compound with a brief duration of action. Estriol is a poor stimulator of uterine growth and plasminogen activator activity in vivo. Chemical modifications of the estriol molecule produce long-acting derivatives that result in a prolonged input of hormone receptor complexes into the nucleus and a prolonged and marked stimulation of uterine growth. In human endometrial cells in primary tissue culture, E₃ has 12% the affinity of estradiol (E₂) for cytosol estrogen receptor and it is quite effective yet slightly less potent than estradiol in stimulation of progesterone receptor synthesis. Low concentrations of E₃(10⁻¹⁰ M) stimulate growth of MCF-7 cells in vitro and dose-response curves show E₃ to be only slightly less effective than E₂. In these endometrial and breast cancer cell systems in vitro, there is no metabolism of E₃ while E₂ is metabolized to estrone.Hence, estriol is an effective estrogen in vitro. In vivo, it is short-acting, but it can be made a full estrogen agonist when given at a sufficiently high concentration or in a chemically modified form which prolongs its activity by enabling effective concentrations of the compound to be maintained in the blood and in target tissues.     

Characterization of the interaction between estrogen metabolites and taurocholate for uptake into isolated hepatocytes. Lack of correlation between cholestasis and inhibition of taurocholate uptake

The cholestasis induced by estrogen metabolites has been postulated to be due to an inhibition of bile acid transport. Therefore, the uptake of [³H]taurocholate (TC) into isolated hepatocytes was examined in the presence of known cholestatic steroid glucuronides. The cholestatic d-ring glucuronide conjugates of estradiol, estriol, ethynylestradiol and dihydrotestosterone did not inhibit the uptake of TC suggesting that these organic anions are transported by different carrier systems. Estrone sulfate inhibited TC uptake 65% but did not decrease bile flow following i.v. administration to the rat (22μ mol/kg), under conditions which the steroid glucuronide estradiol-17β-(β-d-glucuronide) (E₂17G) decreased bile flow 100%. The hepatocytic uptake of [³H]E₂17G (100μM) was inhibited by estriol-16α-(β-d-glucuronide) (200 μM, 40%) and estradiol-17β-3-(β-d-glucuronide) (200 μ M, 22%) as well as by the organic anions bromosulfophthalein (150 μM, 57%), dibromosulfophthalein (150 μM, 59%), indocyanine green (150 μM, 62%), rose bengal (150μM, 56%) and bilirubin (50μM, 40%). Thus, the bile acids and steroid glucuronides are transported into the hepatocyte by different carrier systems so that the cholestasis induced by the steroid d-ring glucuronides cannot be explained by an inhibition of bile acid uptake. Furthermore, the hepatocytic uptake of E₂17G occurs by a carrier system similar to that for the other steroid glucuronides and organic anions.     

Preparation of specific antisera to estrone-3-glucuronide and estriol-3-glucuronide

The preparation and antigenic properties of estrone-3-glucuronide- and estriol-3-glucuronide-bovine serum albumin conjugates in which the hapten is linked to the carrier protein through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Antibodies raised against the two immunogens in the rabbit possessed high specificity to estrone-3-glucuronide and estriol-3-glucuronide, respectively, exhibiting little cross-reactivities with other estrogen conjugates and no cross-reactions with related steroids except for free estrogens, their 3-methyl ethers and 3-sulfates. The cross-reactive antibodies were eliminated by partial immunoadsorption on affinity chromatographic media using the estrone-3-methyl ether 17-(O-carboxymethyl)oxime- and estriol-3-methyl ether 16 (or 17)-hemisuccinate-aminohexyl Sepharose conjugates, respectively. The purified antisera exhibited no cross-reactivities with free estrogens and ring A conjugates of estrone and estriol.     

Sensitive determination of estriol-16-glucuronide using surface plasmon resonance sensing

For the quantitative evaluation of low levels of an estriol metabolite of estriol (estriol-16-glucuronide (E3-16G)) in liquid media, we developed a simple and highly sensitive immunoassay using a surface plasmon resonance (SPR) biosensor which did not require any time-consuming sample pretreatment steps. E3-16G was conjugated to ovalbumin (OVA) through an oligoethylene glycol (OEG) linker to form protein conjugates (E3-16G-OEG-OVA), which were then immobilized on a carboxymethyl dextran-coated sensor chip via amine coupling to develop inhibition immunoassays. A limit of detection (LOD) of 76 pg/mL was achieved using a rabbit anti-sheep primary antibody as a binding agent. The detection limit was further improved by using synthesized gold colloids (15 nm) as high mass labels conjugated to the primary antibody. In this Au nanoparticle-enhanced assay, the concentration of E3-16G in aqueous samples could be determined in 7.5 min at a level as low as 14 pg/mL. In addition, the high stability of the E3-16G-OEG-OVA surface gave no obvious drop in antibody-binding capability after more than 1000 binding/regeneration cycles which significantly lowered the research cost.     

The biosynthesis of estriol-3,16-disulfate by guinea pig liver homogenate

Incubation of ³H-estriol in guinea pig liver homogenate fortified with uridine-5′-diphosphoglucuronic acid afforded a new conjugate in addition to the expected ³H-estriol-3-glucosiduronate. Employing methodology including chromatography on alumina and Celite, high voltage paper electrophoresis and reverse isotope dilution, its structure was established as estriol-3-16-disulfate. The chemical synthesis and some of the physical properties of the disulfate are described.     

Direct radioimmunoassay of specific urinary estrogen glucosiduronates in normal men and nonpregnant women.

Direct radioimmunoassay are described for the measurement of each of three specific estrogen glucosiduronates: estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate and estriol-16 alpha-glucosiduronate in urine. Each assay utilizes a specific antiserum prepared by complexing the carboxylic acid group of the appropriate glucosiduronate to the epsilon-amino group of lysine in bovine serum albumin or bovine thyroglobulin. The antisera showed little or no cross reactivity toward other estrogens that might be present in significant amounts in urine. These antisera were used for the direct assay of the conjugates in urine from normal men and nonpregnant women without prior extraction or chromatography. The values were similar to those obtained after extraction, chromatographic purification on DEAE-Sephadex and subsequent immunoassay; The following mean values +/- SE (microgram/g creatinine) were obtained: estrone glucosiduronate, male 10.1 +/- 0.6, follicular phase female 17.3+/- 1.6, luteal phase female 31.8 +/- 2.5; 17 beta-estradiol-17-glucosiduronate, male 1.7 +/- 0.3, follicular phase female 2.4 +/- 0.1, luteal phase female 4.2 +/- 0.4; estriol-16 alpha-glucosiduronate, male 1.8 +/- 0.2, follicular phase female 4.7 +/- 0.9, luteal phase female 10.0 +/- 1.6.     

Sensitive determination of estriol-16-glucuronide using surface plasmon resonance sensing

For the quantitative evaluation of low levels of an estriol metabolite of estriol (estriol-16-glucuronide (E3-16G)) in liquid media, we developed a simple and highly sensitive immunoassay using a surface plasmon resonance (SPR) biosensor which did not require any time-consuming sample pretreatment steps. E3-16G was conjugated to ovalbumin (OVA) through an oligoethylene glycol (OEG) linker to form protein conjugates (E3-16G-OEG-OVA), which were then immobilized on a carboxymethyl dextran-coated sensor chip via amine coupling to develop inhibition immunoassays. A limit of detection (LOD) of 76 pg/mL was achieved using a rabbit anti-sheep primary antibody as a binding agent. The detection limit was further improved by using synthesized gold colloids (15 nm) as high mass labels conjugated to the primary antibody. In this Au nanoparticle-enhanced assay, the concentration of E3-16G in aqueous samples could be determined in 7.5 min at a level as low as 14 pg/mL. In addition, the high stability of the E3-16G-OEG-OVA surface gave no obvious drop in antibody-binding capability after more than 1000 binding/regeneration cycles which significantly lowered the research cost.     

Specificities of antisera against estrogens linked to albumin at different positions (C6, C11, C16, C17)

Antisera were raised in rabbits using conjugates of albumin with 11-hemisuccinates of 11α-OH E₁ and 11α-OH E2. These antisera were compared with antisera to 6-oxo E₂ 6-CMO-, E₂ 17-succinyl- and E₃ 16, 17-disuccinyl-BSA by radioimmunoassay using a statistically designed three-point assay.Sheep anti-(rabbit γ-globulin) coupled to cellulose was used for the separation of antibody-bound and free labeled hapten.Antisera obtained with haptens linked to the carrier at the 17(16) position poorly discriminated between the various estrogens. Antisera obtained with 6- and 11-conjugates showed a much better specificity. In addition the specificity was influenced by using either estrone, estradiol or estriol as tritiated labels. This gives the possibility to determine different parameters by employing different labeled hormones.     

Relative distribution of estrone,estradiol and estriol between fetal and maternal perfusates during perfusions of human term placentas with labeled C19 precursors

Published results from in vivo experiments carried out in Rhesus monkeys and from in vitro perfusions of human term placentas have indicated that placental estradiol (E₂) is preferentially released towards the mother whereas estrone (E₁) is about evenly distributed between fetal and maternal circulation.In order to examine the distribution of estriol, relative to that of E₁ and E₂, we have now prepared [³H]16-hydroxyandrostenedione by incubation of [6,7-³H]androstenedione with Streptomyces roseochromogenus and perfused placental cotyledons with mixtures of these two labeled precursors. Measurement of the concentrations of tritiated E₁, E₂ and E₃ in the maternal and fetal perfusates, flowing at approx 10 and 5 ml/min, respectively, indicated that the distribution of E₃ is different from that of E₂ and resembles the distribution of E₁.The simple perfusion system being used shows differences in the distribution of various estrogens between fetal and maternal perfusates which may reflect the in vivo situation and offers the opportunity for experimental examination of various explanations for these differences, e.g. existence of specific carrier systems in the syncytial membranes, specific binding of the estrogens to secreted placental proteins, and actions of placental and decidual 17β-hydroxysteroid dehydrogenases.     

Biological responses and histological studies of estriol and estriol sulfate in fetal and newborn uteri of guinea pig

The biological responses of estriol (E₃) and of estriol-3-sulfate (Ea₃-S) in the fetal and newborn uteri of guinea pig were studied. After a treatment of E₃ (1 mg/kg/day) or E₃-S (1.4 mg/kg/day) to pregnant guinea pigs (49–60 days of gestation) for 6 days, both estrogens provoke a significant uterotrophic effect in the fetal uterus which increases in weight 1.8–2.5 times in relation to the non-treated animals. The stimulation of progesterone receptor (PR) is also very intense, 7–12 times in relation to the control animals.In another series of experiments newborn guinea pigs (2-day old) were treated with 100μg/animal of E₃ or 140 μg/animal of E₃-S for a short (2 days) or a long (12 days) period. Concerning the uterotrophic effect, the weight of the uterus increases 1.8–2.5 times (in relation to the non-treated animals) after a 2-day treatment and the effect continues to increase up to 4–5 times in the 12-day treated animals. In opposition to the fetal uterus, the effect on PR provoked by E₃ or E₃-S is very limited (only an increase of 1 time in relation to the non-treated animals); the effect is even less intense after the 12-day treatment. Histological studies show an intense hypertrophic effect particularly in the epithelium of the endometrium in the fetal and newborn uteri for both E₃ and E₃-S. In the newborns the effect is also intense on the epithelium of the uterine gland.It is concluded that: (1) Estriol and estriol sulfate are very active and with similar intensity on the uterine weight before and after birth; (2) The stimulatory effect on PR decreases very significantly after birth and after a long treatment; (3) E₃S can act as a potent hormonal precursor.     

Estriol in human breast cyst fluid

Although cystic lesions of the breast are not precancerous per se, statistical studies have indicated that this condition predisposes a 2- to 4-fold greater risk for breast cancer. Seeking a hormonal etiology to this correlation, investigators have analyzed breast cyst fluid (BCF) for steroids and have compared the levels to those in the blood. The 17-ketosteroids-androsterone, dehydroisoandrosterone and their sulfates are elevated in BCF. The same is true for estrone sulfate and estradiol-3-sulfate. We have found the most dramatic differences with estriol-3-sulfate (E₃-3S), the concentration of which ranged from 187–6134 pg/ml in over 40 specimens analyzed, whereas in 12 serum specimens from normal women, E₃-3S was barely detectable. The origin of E₃-3S is not known. [³H]E₃-3S is not concentrated in BCF following its injection into an arm vein. The blood half-life of [³H]E₃-3S is much lower than that of estrone sulfate. Samples of breast nipple aspirates from normal women were also analyzed for E₃-3S. None could be detected. The best explanation of the data accumulated thus far is that E₃-3S is synthesized at the epithelial lining of the cyst and released into the BCF, from which its efflux is inefficient.     

A role for prostaglandins in the regulation of the placental blood flows

A model is proposed for the regulation of the placental blood flows to the near-term pregnancy. The model has three features. 1) The maternal uterine and fetal placental tissues can synthesize constrictor and dilator prostaglandins. 2) Prostaglandins can cross the placenta. 3) There must exist a prostaglandin which has a vasodilating action in one of the placental circulations and a vasoconstricting action in the other circulation.Evidence is provided to indicate that in the sheep, prostaglandin E₂ (PGE₂) can cross the placenta and has a vasodilating action in the uterine placental circulation and a vasoconstricting action in the umbilical placental circulation.The placenta and the lung are compared and PGE₂ is shown to have similar actions in each of these organs.