Equol: a contributor to enigmatic immunoassay measurements of estrogen

The efficacy of radioimmunoassay (RIA) for the measurement of estradiol-17 beta (E2) in murine plasma was investigated. When Sephadex LH-20 or celite column chromatography was used to separate E2 from estrone (E1) and other cross-reacting compounds, the results were erratic if small volumes of mouse plasma were resolved. Assay of a diethyl ether extract of plasma (500 microL) was the most practical method for estimating the concentration of estradiol-17 beta in mice. This method was used to determine the pattern of estrogen secretion during the estrous cycle, on the day of implantation and during pregnancy. No convincing change in estrogen secretion was observed in the diestrous/proestrous mouse. By comparison, estrogen levels were elevated during pregnancy. Taken together, these results implied that cross-reactive components in plasma masked low levels of endogenous estrogen. Further evaluation of mouse plasma and urine using a co-chromatography technique to examine estrogen elution from a reverse-phase HPLC system followed by GC/MS analysis indicated the presence of equol [7-hydroxy-3-(4-hydroxyphenyl)chroman], a phytoestrogen metabolite with a ring structure similar to estradiol-17 beta. Equol and possibly other cross-reactive components of plasma may account for the apparent lack of increased estrogen secretion during the mouse estrous cycle and on the day of implantation as determined by the radioimmunoassay of ether extracts of plasma.     

Estrogenic action of estriol fatty acid esters

Recent studies suggest that, estriol, like estradiol, is biosynthetically esterified with fatty acids. We have synthesized the stearate estriol, at C-16 alpha, C-17 beta and the diester, C-16 alpha,17 beta and tested these D-ring esters for their estrogenic action both in vivo and in vitro, comparing them to estradiol, estriol and estradiol-17-stearate. None of the estriol esters bind to the estrogen receptor. They are only weakly estrogenic in a microtiter plate estrogen bioassay: stimulation of alkaline phosphatase in the Ishikawa endometrial cells. However, both estriol monoesters are extremely potent estrogens when injected subcutaneously (in aqueous alcohol) into ovariectomized mice. Compared to the free steroids, they produced a dramatically increased uterine weight with a greatly prolonged duration of stimulation. The 16 alpha,17 beta-diester also induced a protracted uterotrophic response, but the stimulation of uterine weight was comparatively low. Since the esters of estradiol and estriol do not bind to the estrogen receptor, their estrogenic signal must be generated through the action of esterase enzymes. These naturally occurring esters have the potential of being extremely useful pharmacological agents for long-lived estrogenic stimulation.     

Radioautographic study of the effect of estradiol-17 , estrone, estriol, progesterone, testosterone and corticosterone on the in vitro uptake of 2,4,6,7- 3 H estradiol-17 by uterine eosinophils of the rat

The in vitro uptake of 2,4,6,7-tritiated estradiol-17beta in uterine eosinophils of the rat was inhibited by the presence of nonradioactive estradiol-17beta, estrone, and estriol, but not by progesterone, testosterone, or corticosterone. This action is attributed to competition between tritiated estradiol and the various estrogenic compounds for the same binding site. Compounds without any estrogenic activity do not compete. The proposal is made that the eosinophil binding system and the 8S-5S binding system are involved in different mechanisms of estrogen action. The parallelism between the doses of estradiol and estriol needed to promote certain estrogenic early effects in the uterus, and the affinity of these steroids for the eosinophil uptake sites, suggests that uterine eosinophils might be responsible for some of these early effects, such as water imbibition, histamine releasing activity, and estrogen priming effect.     

Changes in estrogen levels in maternal venous plasma after intra-amniotic infusion of prostaglandin F2 for therapeutic abortion

Peripheral plasma levels of estrone, estradiol-17beta and estriol were measured by the method of Shutt and Cox in 10 women following intra-amniotic infusion of prostaglandin F2alpha (PGF2a) for therapeutic abortion. Initial dose was 30 mg, followed if necessary, by doses of 15 mg at 24 hours and 42 hours. Gestational age of pregnancies ranged from 14 to 19 weeks, with a mean of 16 weeks. All 10 women completely aborted. Mean induction-abortion interval was 24 + or - 12 hours. The mean estrone, estradiol 17beta and estriol levels declined to about half of the pre-infusion levels after 80% of the induction-abortion interval had elapsed. The main decline in estrogen levels occurred in individual women either during the 1st quarter or during the last quarter of the induction-abortion interval. There were no significant relationships between changes in estrogen levels and the interval from 1st administration of PGF2a to subsequent abortion.     

Effects of 17-beta estradiol and estriol on NMDA-induced toxicity and apoptosis in primary cultures of rat cortical neurons.

Estrogens possess neuroprotective and antiapoptotic properties, however, the issue of involvement of estrogen receptors (ER)-dependent genomic pathway in these effects still remains controversial. Moreover, the majority of data on antiapoptotic effects of estrogens concern non-neuronal cells. In the present study we compared effects of the potent ER agonist, estradiol-17beta (E2), and its metabolite with a weak affinity for ER, estriol, on the neurotoxicity induced by high (1 and 5 mM) NMDA concentrations and on the apoptosis induced by low (0.1 mM) concentration of NMDA in rat primary cortical neurons. The obtained data showed that 24-hour exposure of cortical neurons to NMDA (0.1-5 mM) resulted in a dose-dependent increase in LDH level. Twenty four-hour pretreatment with estriol (100 nM and 500 nM) reduced the NMDA (1 and 5 mM)-induced toxicity by 16-26%, while estradiol-17beta (500 nM) reduced NMDA (5 mM)- induced toxicity by 14%. Twenty four hour exposure of cortical neurons to NMDA (0.1 mM) resulted in decrease of the level of antiapoptotic protein - Bcl-2 by 60% and increased the number of apoptotic cells by 50% compared to the control. Twenty four hour pretreatment with estradiol-17beta or estriol (100 and 1000 nM) prevented the NMDA-induced apoptotic changes. The specific estrogen receptor antagonist ICI 182,780 (100 nM) had no effect alone and did not antagonize the effects of estrogens on NMDA-induced toxicity as well as on changes in Bcl-2 level. The higher efficacy of estriol, together with the fact that the specific ER receptor antagonist, ICI 182,780, did not inhibit the above-described effects support the hypothesis about a nongenomic mechanism of the anti-NMDA action of estrogens.     

Mass fragmentographic determination of eleven estrogens in the body fluids of pregnant and nonpregnant subjects

Mass fragmentographic determinations of 11 estrogens in urine, bile, or plasma of pregnant and nonpregnant subjects were made. The estrogens (estriol, estrone, 2-methoxyestrone, estradiol-17beta, estradiol-17alpha, 16-epiestriol, 17-epiestriol, 16-alpha-hydroxyestrone, 16beta-hydroxyestrone, 16oxoestradiol-17beta, and 15alpha-hydroxyestrone) were quantitatively determined in bile from 1 male and 3 postmenopausal women, in the urine of a nonpregnant woman, and in a 20 ml pool of late pregnancy plasma obtained from 10 women. The specificity of mass fragmentography as compared with gas chromatography is considered better because a characteristic ion is monitored rather than the total ion current measured by flame ionization detection and reliable measurements can be made in the presence of larger amounts of impurities, resulting in a shortened fractionation procedure.     

Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells

The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M androstenedione, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of 4 X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge.     

Chemical and biological evidence for presence of estrogens in the domestic turkey (Meleagris gallopavo)

Solvent partitioning, column chromatography on activated alumina, and Ittrich extraction of Kober chromogens were employed to isolate, identify and quantitate estrone, estradiol-17B and estriol from blood of individual turkey hens. The intravaginal tetrazolium method was used to estimate the biological potencies of the three fractions separated chromatographically. The data show that estrone is the main estrogen present in heart blood of laying turkeys, with lesser amounts of estradiol-17B and estriol. Data are presented on the accuracy, precision, sensitivity, and specificity of the methodology used.     

Estradiol levels near the time of ovulation in the bonnet monkey (Macaca radiata)

Plasma estradiol-17beta and total progestins were determined to delineate the relationship between preovulatory estradiol-17beta peak and ovulation in the bonnel monkey (Macaca radiata). 6 monkeys were studied for 15 menstrual cycles. In subsequent cycles, serial laparotomy was performed in 5 of the 6 monkeys to correlate ovarian morphology to plasma estradiol-17beta. In 11 of the 15 cycles, estradiol-17beta peaks were 3- to 7-fold above baseline levels near the time of expected ovulation (Cycle Days 7-12). Plasma progestin rose significantly from follicular phase levels of .5 ng/ml to 2.6 ng/ml the day of the estradiol-17beta peak with peak levels of 4.5 ng/ml on the following day. Ovarian morphology in 4 of the 5 observed by laparotomy demonstrated ovulation within 48 hours following an estradiol-17beta peak of approximately 300 pb/ml.     

Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers

The binding of catechol estrogens, epoxyenones and methoxyestrogens was evaluated using estrogen receptors in cytosol prepared from human breast cancers. The relative affinity of 2-hydroxyestradiol, a metabolite formed in vitro from estradiol-17 beta by breast cancer cells, was indistinguishable from that of estradiol-17 beta. 4-Hydroxyestradiol, which is also a metabolite of estradiol-17 beta, associated with the estrogen receptor with a relative affinity approximately 1.5-fold greater than that of estradiol-17 beta. Epoxyenones and methoxyestrogens were weak competitors compared to the binding of estradiol-17 beta, exhibiting relative affinities 3% or less than the affinity of estradiol-17 beta. Sucrose density gradient centrifugation revealed that both 2- and 4-hydroxyestradiol inhibited the binding of estradiol-17 beta to both the 4S and 8S isoforms of the estrogen receptor in a competitive manner, with a Ki = 0.94 nM for 2-hydroxyestradiol and a Ki = 0.48 nM for 4-hydroxyestradiol. It can be concluded that these data demonstrate a specific receptor-mediated estrogenic action for both of these catechol estrogens.     

Competitive protein-binding assay of estrone, estradiol-17 or estradiol-17 in human or ruminant plasma

A procedure for the assay of estrone, estradiol-17β or estradiol-17α in plasma is described. The technique also appears applicable to the assay of estriol in plasma. The procedure uses a semi-automatic extraction of plasma, rapid micro-alumina column chromatography and competitive binding of the estrogens to stable proteins of sheep uterine cytosol. The use of alumina column chromatography results in consistently low blanks. The assay has been evaluated for the measurement of estradiol-17β and estrone in human and sheep plasma, and for estradiol-17α and estrone in goat plasma. The change in binding affinity of estradiol-17α relative to estradiol-17β when incubated in sheep uterine cytosol at two different temperatures (25°C and 4°C), makes it possible to differentiate the two epimers of estradiol. Measurement of estradiol-17β down to 10 pg and of estrone and estradiol-17α to 25 pg are maintained in routine analyses. The specificity of the procedure was thoroughly checked by various methods, including comparison with spectrophotofluorimetric analysis.     

Structure-activity relationships of estrogens. Effects of 14-dehydrogenation and axial methyl groups at C-7, C-9 and C-11

Thirty compounds were evaluated in the rat for uterotropic effects, inhibition of gonadotropin release, and competitive displacement of (3H) estradiol-17 beta from uterine cytosolic preparations. 7 alpha-Methylestradiol-17 beta was 150% as active as estradiol-17 beta as an uterotropic agent. Estradiol-17 beta was the most active inhibitor of gonadotropin release. 11 beta-Methylestradiol-17 beta had 124% of the activity of estradiol-17 beta in displacing (3H) estradiol-17 beta from the "estrogen receptor." The 9 alpha-methyl group considerably decreased the potency of estrogens in any of the three assays. The 14-dehydro modification was advantageous only in the estradiol-17 beta 3-methyl ether series. Uterotropic activities and inhibition of gonadotropin release did not parallel. The best compound for inhibiting gonadotropin release, as compared to uterotropic activity, was estrone. The "estrogen receptor" assay data correlated fairly well with uterotropic assay data, but only for compounds having free 3-hydroxyl groups; even so, some exceptions were noted.     

Structure-activity relationships of estrogens: effects of esterification of the 11 beta-hydroxyl group

Fourteen esters (formate, acetate, propionate, butyrate, hexanoate, heptanoate, and benzoate) located at C-11 of 11 beta-hydroxyesterone and 11 beta-hydroxyestradiol-17 beta were synthesized and evaluated for uterotropic and gonadotropin release inhibition in rats, as well as their ability to displace (3H) estradiol-17 beta from the rat uterine cytosolic estrogen receptor. The most potent uterotropic agent was 11 beta-formoxyestrone which was 1,625 or 2,500 times as active as 11 beta-hydroxyesterone in the uterotropic or gonadotropin release inhibition assay, respectively. 11 beta-Formoxyestrone was 7.5 times as uterotropic as estradiol-17 beta and equal to estradiol-17 beta in inhibiting gonadotropin release. However, the most potent inhibitor of gonadotropin release was 11 beta-acetoxy-estradiol-17 beta which had 133% of the activity of estradiol-17 beta, although it had only 38% of the activity of estradiol-17 beta in the uterotropic assay. Esters larger than the acetoxy group showed sharply decreased activities in either assay. Despite the high estrogenic potency of the 11-formates or 11-acetates, they were rather weak (6% to 35% as active as estradiol-17 beta) in displacing (3H) estradiol-17 beta from the rat uterine cytosolic estrogen receptor.     

The estrogenic activity of certain phytoestrogens in the Siberian sturgeon Acipenser baeri

Various phytoestrogens such as formononetin, daidzein, genistein and equol were synthesized. Their purity was assessed by various analytical techniques including melting point determination, thin-layer chromatography (TLC), infra-red spectra (i.r. spectra), nuclear magnetic resonance (1H- and 13C-NMR) and gas chromatography coupled with mass spectrometry (GC-MS). The estrogenic activity of these compounds, as well as biochanin A and coumestrol, was biologically tested by the induction of vitellogenin secretion in yearling sturgeon and compared to the activity of estradiol-17 beta. Pure daidzein, biochanin A, genistein, equol and coumestrol all had estrogenic activity as assessed by their induction of hepatic synthesis of vitellogenin when administrated intraperitoneally to yearling Siberian sturgeon. Coumestrol seemed to be the most potent compound, inducing the most vitellogenin secretion with the lowest dose administered. Formononetin was inactive when administered by the intraperitoneal route. All the phytoestrogens tested were considerably less potent than estradiol-17 beta.     

Gap junction modulation in rat uterus. III. Structure-activity relationships of estrogen receptor-binding ligands on myometrial and serosal cells

A number of steroidal and nonsteroidal estrogen receptor-binding ligands were tested for their ability to affect the formation and internalization of gap junctions in hypophysectomized rat uterine myometrial and serosal cells. Potent estrogen, including diethylstilbestrol, estradiol benzoate (EB), estradiol-17 beta, and the weak estrogens, estriol and estrone, stimulate formation of macular and annular gap junctions in myometrium in a dose-dependent fashion when administered in daily injections over 5 days. Induction of annular gap junctions in the uterine serosal epithelium follows a similar dose-dependent pattern of estrogen stimulation but requires lower levels of hormone to initiate the response. In myometrium, differential stimulation of circular and longitudinal myometrial cell layers was observed, with 3 to 5 times more gap junctions detected in the circular than in the longitudinal layer. Progesterone, estriol, or estrone suppress the myometrial gap junction response to EB when administered concurrently with EB. However, the EB-stimulated appearance of myometrial cell gap junctions was blocked when the progesterone-to-estrogen ratio exceeded 100:1. The estrogen receptor-binding androgens, 5 alpha-androstane-3 beta,17 beta-diol (Adiol) and delta 5-androstene-3 beta,17 beta-diol failed to induce myometrial gap junctions at doses up to 5 mg/day for 5 days, whereas Adiol did induce annular gap junctions in the serosal cells at the highest dosage tested. Of the triphenylethylene derivatives and related compounds evaluated, including mixed isomers of tamoxifen and CI 628, the cis (zuclomiphene, ZUC) and trans (enclomiphene) isomers of clomiphene citrate, and a fixed-ring antiestrogen, nafoxidine, only ZUC was able to induce gap junctions in myometrial and serosal cells. These studies indicate that induction of gap junctions in rat uterine myometrial cells is an estrogen-dependent response that requires higher levels of estrogen than other estrogen-dependent target cell responses in the rodent uterus.     

Ultrastructural changes in guinea pig endometrial cells during the estrous cycle.

In the guinea pig, the estrous cycle is characterized by a constant measurable level of plasma progesterone with two peaks: the first one associated with the peak of plasma estradiol-17 beta occurring at proestrus and the second, during diestrus, more pronounced at the time at which the level of estradiol-17 beta is undetectable. The progesterone receptor content is the highest on day 1 and the lowest on day 10 of the estrous cycle, which lasts 16.3 +/- 1.5 days (n = 37; mean +/- SD). There is a positive correlation between the plasma level of estradiol-17 beta and the progesterone receptors detected immunocytochemically in both endometrial epithelial and stromal cells. The general morphology of the endometrium during proestrus and estrus is consistent with an estrogenic stimulation, i.e., a smooth and regular surface of the endometrium and the presence of numerous microvilli on the cell surface. However, a moderate secretory activity also occurs in proestrus and estrus. During postestrus, the glandular cells display an increase in characteristic secretory features which parallels the rise of progesterone in the plasma.     

Isolation and characterization of an estrogen binding protein which may integrate the plethora of estrogenic actions in non-reproductive organs

A putative estrogen receptor (pER) from mouse liver has been characterized. The heterodimer protein (81–84 kDa) consists of two covalently bound subunits (61–67 and 17–27 kDa) with following characteristics: sedimentation constant — 4.9 S; IP — 4.8; dissociation constant (K_d) for estradiol-17β binding — 0.7 nmol; binding sites — 0.746 pmol/mg protein; relative binding affinity — estradiol-17β — 100, estrone — 80 and estriol — 30; specificity — does not bind, other natural steroids, synthetic estrogens, antiestrogens and bioflavonoids. Importantly, immunosuppressants, neuroleptic and carcinogens influence ³H-estradiol-17β binding to pER. Interestingly, pER is a serine phosphatase and this may have relevancy to estrogen action in Alzheimer's disease. The polyclonal anti-pER antibody does not react with estrogen receptors (ER). ER antibody does not react with pER. Remarkably, anti-pER antibody reacts with calcineurin, a brain phosphatase and anti-calcineurin antibody reacts with pER. Immunohistochemical analyses showed that pER is undetectable in reproductive organs (except ovary). It is localized on the plasma or the nuclear membranes in some, in cytoplasm and/or nucleus in other cells of non-reproductive organs (skeletal, neural, vascular, hair and retina), and in tumors (mammary, endometrial and prostate cancers, and prostatic hyperplasia). The information presented justifies the proposition that pER may mediate the estrogenic actions in non-reproductive organs.     

Opposing effects of androgen and estrogen on pituitary-adrenal function in nonpregnant primates

Maternal administration of androstenedione produces a sustained fall in maternal plasma adrenocorticotropic hormone (ACTH) concentrations in the pregnant nonhuman primate. We hypothesize a negative feedback influence on the maternal hypothalamo-pituitary-adrenal (HPA) axis by androgens in primates. This may reflect an important maternal adaptation during pregnancy in primates preventing premature induction of labor by maternal stress. However, androstenedione is precursor for placental estradiol-17beta synthesis, and infusion of androstenedione into pregnant primates elevates maternal plasma estradiol-17beta to term concentrations. Thus, it could be argued that 1) the effects attributed to androstenedione on the maternal HPA axis are mediated by estrogen rather than by androgen and 2) the negative influence of androgens may be on placental ACTH rather than, or in addition to, pituitary ACTH. To discriminate between androgenic and estrogenic effects of androstenedione on pituitary and/or placental ACTH function in primates we measured plasma ACTH, cortisol, and dehydroepiandrosterone sulfate (DHEAS) concentrations in nonpregnant baboons after treatment with either androstenedione or estradiol-17beta. Nine female baboons were studied between 14 and 22 days postpartum prior to estrous cycling. After 2 days of baseline, a continuous i.v. infusion of androstenedione (1.5 mg/kg per h in 10% intralipid, IL) was started at 0900 h and maintained for 9 days in 3 baboons. A similar protocol was carried out in another 3 baboons that received a continuous i.v. infusion of estradiol-17beta (10 microg/kg per h in 10% IL) instead of androstenedione. Three additional baboons received continuous i.v. IL vehicle alone and served as controls. Arterial blood samples (0.5 ml) for measurement of plasma hormones were taken during baseline and after 1, 3, 5, 7, and 9 days of infusion. Baseline plasma ACTH, DHEAS, and cortisol concentrations were similar among all groups. Plasma ACTH did not change during IL, increased following estradiol-17beta, and fell during androstenedione treatment. Accordingly, plasma cortisol and DHEAS concentrations were also unaltered by IL, and both steroids increased during estradiol-17beta treatment. In contrast, plasma cortisol and DHEAS remained unaltered from baseline during androstenedione treatment, despite the fall in plasma ACTH measured at this time. These data in the nonpregnant baboon 1) are consistent with negative feedback on pituitary ACTH by androgens and 2) demonstrate a positive influence on pituitary-adrenal function by estrogen in primates.     

Identification of phytoestrogens in the urine of male dogs

It is becoming increasingly apparent that dietary factors may play a role in the etiology of hormone dependent neoplasias. It has been hypothesized that estrogens play some role in the etiology of benign prostatic hyperplasia (BPH) in the canine. The presence of estrogen receptor binding activity in a fraction of canine urine purified by high performance liquid chromatography (HPLC) that did not correspond to estriol, estradiol, estrone or any of their primary metabolites was observed in the present study. We used thermospray-mass spectrometry and GC-MS to identify the phytoestrogens daidzein, equol, formononetin and genistein in HPLC purified fractions of urine obtained from male beagles. Using the same techniques we also confirmed the presence of daidzein and genistein in the commercial diet fed to these same dogs. Using the immature rat uterine cytosol estrogen receptor assay, relative binding affinities of 0.08, 1.1, less than 0.01 and 3.9% were obtained for daidzein, equol, formononetin and genistein, respectively when compared to estradiol (100%). In conclusion, phytoestrogens are present in urine of male beagles. Moreover, the commercial diet fed to these dogs contains isoflavones which can be converted to equol by intestinal microflora. These results suggest the need for investigations of phytoestrogens (e.g. equol) excreted into the urine daily and its relationship to the incidence and severity of BPH in the dog.     

Comparative specificity of antisera raised against estrone, estradiol-17 and estriol using 6-0-carboxy-methyloxime bovine serum albumin derivatives

Antisera for the radioimmunoassay of estrone and estradiol-17β in plasma are usually raised against estradiol-17β coupled to a protein through a derivative at carbon 17. Such antisera cross react with other naturally occurring estrogens, necessitating preliminary chromatographic separation. This difficulty could be overcome by the use of more specific antisera. We have raised antisera against the 6-0-carboxymethyloxime-bovine serum albumin (BSA) derivatives of estrone, estradiol-17β and estriol respectively. We have determined cross reactions with a number of estrogens and other naturally occurring steroids, and have compared the cross reactivity with that of an antiserum raised against estradiol-17β-17-succinyl-BSA. The former antisera show greatly reduced cross reaction with naturally occurring estrogens known or thought to be in relatively high concentration in plasma, as compared with the latter antiserum, but at the expense of greatly increased cross reaction with estrogens substituted at carbon 6. However, since these latter estrogens are thought to be in low concentration in plasma, the use of antisera raised against the 6-0-carboxymethyloxime-BSA derivatives should result in a net gain in specificity. The antisera raised against the estrone and estriol 6-0-carboxymethyloxime-BSA derivatives should be particularly useful.