An enzyme-immunoassay for estriol.

A practical method was developed for enzyme-immunoassay of serum estriol, with alkaline-phosphatase as a marker enzyme. Alkaline-phosphatase was conjugated with estriol-6-(O-carboxymethyl) oxime using water soluble carbodiimide. The estriol-alkaline-phosphatase complex, which has both enzyme activity and capacity to bind anti-estriol serum, was obtained by Sephadex G-200 gel filtration. This complex, which was stable for at least 3 months at 4 degrees C, was used as enzyme-labelled estriol. Anti-estriol serum raised against estriol-6-(O-carboxymethyl) oxime bovine serum albumin was employed. "Bound and free" estriol were separated by the double antibody method. A linear relation was obtained between estriol concentration and antibody-bound alkaline-phosphatase activity in the range of 0.2-100 ng estriol/ml. In this assay system, cross-reactivity with other steroids was negligible under physiological conditions, and endogenous alkaline-phosphatase, which increases during the late pregnancy, caused no interference. The coefficients of variation were 3.3-14.2% (within assays), and less than 22% (between assays), and the mean recovery rate was 77.5%. Serum estriol values determined by the present method correlated well with those determined by radioimmunoassay (r=0.90 for total estriol; r=0.98 for free estriol). The present method of enzyme-immunoassay is suitable for measurement of serum estriol during pregnancy.     

THE BIOGENESIS OF PRIMARY SEX HORMONES : I. THE FATE OF ESTRINS INJECTED INTO THE RABBIT

1. A method is given for the extraction and fractionation of rabbit urines which frees these urines of inactive chromogens but permits a quantitative recovery of estrone and estriol for the colorimetric determination of these compounds. 2. Estrone and estriol content of rabbit urine extracts can be determined by the concentration of the colored compound they form upon diazotization with sulfanilic acid and by the modified phenolsulfonic acid test of Cohen and Marrian. Estriol can be determined by the specific reaction first described by David. The technique for these tests is presented. 3. Estriol (300 micrograms) injected into rabbits (a) in heat, (b) pregnant, (c) pseudopregnant, (d) hysterectomized in heat, (e) hysterectomized pseudopregnant, (f) ovariectomized, is excreted in the urine as estriol. Rabbit does in the luteal phase (b, c, and e) excrete 3 to 4 times the amount of estriol excreted by females without corpora lutea (a, d, and f). 4. When estrone (300 micrograms) is injected into the same types of rabbit does types a, b, and c excrete both estrone and estriol, type f excretes both estrone and estriol shortly after ovariectomy, but only estrone at 2 months after castration. Hysterectomized animals (types d and e) never excrete estriol after estrone injection. The total urinary estrin (estrone plus estriol) in estrone-injected animals is increased 2 to 3 times in animals in the luteal phase (b, c, and e). 5. It is concluded that the uterus is the site of conversion of estrone to estriol, and that the conversion cannot take place in a uterus completely free of ovarian control (e.g., in long time ovariectomized animals). 6. In neither estrone-injected nor estriol-injected females is all the injected hormone recovered in the urine. The maximum recovery is 66 per cent. When estrone-benzoate (600 micrograms) is injected 94–98 per cent of the hormone is recovered from animals in the luteal phase (types c and e) and about 79 per cent in an ovariectomized female (type f). These data are taken to indicate that luteal secretions give partial protection against destruction to the hormones. 7. The observation that in certain of the urine extracts the hormone titer by bioassay is somewhat higher than the colorimetric titer may indicate that there is a slight conversion of estrone to estradiol, particularly since no equilenin was found in any of the extracts by colorimetric test. 8. The simultaneous injection of 300 micrograms of estrone and 500 micrograms of progesterone 4 days after an initial injection of 300 micrograms of estrone results in: (1) an increased estrin excretion in females in heat, hysterectomized unmated, and ovariectomized, and a slight decrease in the pseudopregnant female; (2) the appearance of estriol in the urine of the long time ovariectomized animal with no urinary estriol in a control ovariectomized animal receiving no progesterone. These findings are taken to prove that the conversion of estrone to estriol occurs in the uterus under the influence of progesterone. Since animals in heat produce small amounts of estriol after estrone injection it is inferred that the ovaries of estrus rabbits produce small amounts of corpus luteum hormone in the absence of formed corpora lutea.     

A sephadex column radioimmunoassay of unconjugated estriol in pregnancy plasma

A rapid, precise, and accurate radioimmunoassay for unconjugated estriol in pregnancy plasma has been developed which makes use of the adsorptive properties of Sephadex. The estriol is extracted from plasma by adsorption onto a small column of Sephadex. After the proteins and other interfering materials are washed away, the estriol is equilibrated with a limiting amount of specific antibody. The Sephadex column serves as a means of separating the bound from the unbound estriol, the ratio of which is determined by adding to the system tracer amounts of tritium labeled hormone. The sensitivity is about 220 pg in the sample. The intra- and inter-assay coefficient of variation is 5–6%. The method correlated well with one which involved purification of the estriol prior to quantitationby radioiommunoassay.     

Elisa for salivary and plasma estriol in pregnancy

A competitive, sensitive, and rapid enzyme-linked immunoadsorbent assay (ELISA) was developed for the determination of estriol in saliva and in plasma. Horseradish peroxidase (HRP) was used as the label enzyme; separation between free and bound steroid was carried out by insolubilized antibody prepared by adsorbing purified IgG of rabbit anti-6-oxoestriol-6-(0-carboxymethyl)oxime-BSA on polystyrene balls. The enzyme activity was measured by a colorimetric reaction using o-phenylenediamine dihydrochloride and hydrogen peroxide as substrate. The sensitivity of the assay was 12 pg/tube.In order to compare ELISA to RIA estriol estimations in different biological fluids, we selected six women during normal pregnancy, from the 30th to the 40th week of gestation. Salivary estriol was assayed by direct and extraction methods, while the corresponding plasma samples of the same subjects were analyzed only for unconjugated estriol by an extraction method.A good agreement was found between the results obtained by RIA and ELISA: r=0.897, p <0.001 between direct RIA and direct ELISA in saliva; r=0.909, p < 0.001 between extraction RIA and direct ELISA in saliva; and r=0.916, p < 0.001 between extraction RIA and extraction ELISA in plasma. A good correlation (r=0.793, p<0.001) was present between plasma samples by RIA and saliva samples by ELISA (direct method).These results indicate that: 1. ELISA is a reliable method for the determination of estriol in plasma and saliva. 2. Saliva samples can be used for the assay of estriol and therefore for the assessment of fetal conditions during pregnancy.     

The biological role of estriol]

It was demonstrated on the uteri of women and guinea pigs that estriol (in vitro) possessed a marked affinity to the estradiol-binding system of human and guinea pig uteri; the activity of steroid-receptor interaction of estriol in vitro constituted 9.4% for guinea pigs and 17% for man in relation to the estradiol activity. Administration of estriol to guinea pigs in vivo in a dose of 0.25-0.5 mg led to a sharp reduction of the estradiol-binding capacity of the receptor system of the uterus. It is supposed that there existed a competitive relationship between estradiol and estriol for binding with the active centres of the receptor proteins of the uterus.     

Changes in plasma estriol and progesterone during labor induced with prostaglandin F2α or oxytocin

In a double blind study, 12 women received oxytocin for the induction of labor at term and 20 subjects received prostaglandin F_2α (PGF_2α). During the infusions, plasma progesterone and estriol levels were measured, and compared with pre-infusion levels of these steroids. From the analysis of the data, it is concluded that neither the infusion of oxytocin or PGF_2α per se alters the plasma levels of either progesterone or estriol in term pregnant subjects.     

A comparative study of the effect of 17β-estradiol and estriol on peripheral pain behavior in rats

Although estradiol has been reported to influence pain sensitivity, the role of estriol (an estradiol metabolite and another widely used female sex hormone) remains unclear. In this study, pain behavior tests, whole-cell patch clamp recording and Western blotting were used to determine whether estriol plays a role in pain signal transduction and transmission. Either systemic or local administration of 17β-estradiol produced a significant rise of mechanical pain threshold, while estriol lacked this effect in normal and ovariectomized (OVX) rats following estriol replacement. Local administration of 17β-estradiol or estriol significantly decreased ATP-induced spontaneous hind-paw withdrawal duration (PWD), which was blocked by an estrogen receptor antagonist, ICI 182, 780. However, systemic application of estriol in normal or OVX rats lacked this similar effect. In cultured dorsal root ganglion neurons, estriol attenuated α,β-methylene ATP-induced transient currents which were blocked by ICI 182, 780. In complete Freund's adjuvant treated (CFA) rats, systemic application of 17β-estradiol or estriol decreased the mechanical pain threshold significantly, but did not change the inflammatory process. Similar effects were observed after estriol replacement in OVX rats. The expression of c-fos in lumbosacral spinal cord dorsal horn (SCDH) was increased significantly by administration of 17β-estradiol but not estriol, and not by estriol replacement in OVX rats. These results suggest that 17β-estradiol but not estriol plays an anti-hyperalgesic role in physiological pain. However, both peripheral 17β-estradiol and estriol play anti-hyperalgesic roles in ATP-induced inflammatory pain. Systemic application of estriol as well as 17β-estradiol plays hyperalgesic roles in CFA-induced chronic pain.     

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.     

Blood production rates of estrogens in women with differing ratios of urinary estrogen conjugates.

On the basis of the ratios of the estrogen conjugates in their urine (estriol/estrone + estradiol: E3/[E1+E2]), 19 women were divided into two groups: 9 women had ratios less than 0.6 and 10 women had ratios greater than 1.3. All women had measurements made of endogenous estrogens in their plasma by radioimmunoassay. They were then given constant infusions of 3H-estrone, 3H-estradiol and 14C-estriol during days 5-7 and days 20-22 of their cycles, and metabolic clearance rates (MCR) and blood production rates (PB) of estrone, estradiol and estriol were determined. Despite the wide disparity in their ratios of urinary estrogens, no differences could be found between the groups for the MCR's and PB's for all estrogens at either time of the cycle. The mean ratios of PB's (PB3/[PB2+PB1]) of estrone, estradiol and estriol ranged from 0.07 to 0.10 for each group during the cycle. The amounts of estriol entering the blood are small compared to the amounts of estrone and estradiol and do not correlate with the ratios of their urinary conjugates.     

Salivary unconjugated estriol levels in normal third trimester pregnancy - direct correlation with serum levels

A radioimmunoassay for salivary unconjugated estriol concentration during the third trimester of normal pregnancy is described. The performance characteristics of the method were established by determining the non-specific binding, the blank value of the endogenous estriol free ("stripped") saliva, the recovery experiment and intra- and interassay coefficient of the variations. The corresponding serum samples were also analyzed by the same method. An excellent correlation was found between salivary and serum estriol concentrations.     

A rapid specific radioimmunoassay for unconjugated estriol in plasma.

A rapid, non-chromatographic radioimmunossaay for unconjugated estriol in pregnancy plasma has been developed which utilizes a commonly available antiestrogen antisera. Estradiol-17beta and estrone demonstrate 135% relative cross-reactivity with our antiserum, as compared with 100% for estriol. Specificity is achieved by purification of estriol with solvent partitioning using benzene: petroleum ether (1:1). The results obtained using this method are similar to a radioimmunoassay utilizing a highly specific, but commercially unavailable, antiestriol antiserum. The method is precise, with coefficients of variation ranging from 3.0 to 8.2%.     

Immune modulation in multiple sclerosis patients treated with the pregnancy hormone estriol

The protective effect of pregnancy on putative Th1-mediated autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis, is associated with a Th1 to Th2 immune shift during pregnancy. The hormone estriol increases during pregnancy and has been shown to ameliorate experimental autoimmune encephalomyelitis and collagen-induced arthritis. In addition, estrogens induce cytokine changes consistent with a Th1 to Th2 shift when administered in vitro to human immune cells and in vivo to mice. In a pilot trial, oral estriol treatment of relapsing remitting multiple sclerosis patients caused significant decreases in enhancing lesions on brain magnetic resonance imaging. Here, the immunomodulatory effects of oral estriol therapy were assessed. PBMCs collected longitudinally during the trial were stimulated with mitogens, recall Ags, and glatiramer acetate. Cytokine profiles of stimulated PBMCs were determined by intracellular cytokine staining (IL-5, IL-10, IL-12 p40, TNF-alpha, and IFN-gamma) and cytometric bead array (IL-2, IL-4, IL-5, IL-10, TNF-alpha, and IFN-gamma). Significantly increased levels of IL-5 and IL-10 and decreased TNF-alpha were observed in stimulated PBMC isolated during estriol treatment. These changes in cytokines correlated with reductions of enhancing lesions on magnetic resonance imaging in relapsing remitting multiple sclerosis. The increase in IL-5 was primarily due to an increase in CD4(+) and CD8(+) T cells, the increase in IL-10 was primarily due to an increase in CD64(+) monocytes/macrophages with some effect in T cells, while the decrease in TNF-alpha was primarily due to a decrease in CD8(+) T cells. Further study of oral estriol therapy is warranted in Th1-mediated autoimmune diseases with known improvement during pregnancy.     

Estriol concentrations in plasma of normal, non-pregnant women.

Using a rabbit antisera directed against estriol-3-0-carboxy methyl ether complexed to BSA, an immunoassay for estriol (1) was developed. The mean plus or minus SE concentration of estriol in 18 women in days 5-7 of their cycle was 7.9 plus or minus 0.6 pg/ml which was significantly (P less than 0.01) less than the mean value of 11.1 plus or minus 0.8 pg/ml in 15 women in days 20-22 of the cycle. In 3 of 6 women in whom plasma samples were drawn frequently during their cycle, an estriol peak occurred coincident with the estradiol peak. In 3 women from whom plasma was obtained several times during the course of a day estriol levels did not appear to vary significantly. In 8 women who were on oral contraceptives the mean level of estriol was 7.6 plus or minus 1.5 pg/ml. In 8 post-menopausal women the mean level was 6.0 plus or minus 1.2 pg/ml which is significantly (P less than 0.01) less than the mean luteal phase value but not less (P greater than 0.1) than the follicular phase or oral contraceptive user values. We conclude that some of the circulating estriol is directly secreted by the ovary of normal women.     

Estrogen determination using liquid chromatography with precolumn fluorescence labeling

A liquid chromatography procedure is described for the determination of some estrogens using fluorescence detection. The estrogens are labeled by precolumn derivatization with 5-dimethylaminonaphthalene-1-sulfonylchloride (dansyl chloride) and chromatographed on a reversed-phase, C-18 column with a mobile phase consisting of methanol, water, and acetic acid. The eluted analytes are measured with a fluorescence detector using excitation and emission wavelengths of 350 and 540 nm, respectively. The chief advantage of this new procedure is its sensitivity, requiring smaller amounts of sample to detect and quantitate estrogens in biological materials. We could detect less than 400 pg of estriol. With our procedure, this corresponded to about 25 ng in the final reaction mixture, before derivatization. The use of smaller sample volumes could improve this limit. Linearity for dansylated estriol, estrone, and estradiol was excellent over the estrogen range below 100 μg in the sample. This corresponds to approximately 1.7 μg on the column. Within-run precision was better than 5% for the full extraction and derivatization procedure for estriol from pregnancy urine samples. Chromatography is complete within 10 min for dansylated estriol, estrone, and estradiol.     

Direct determination of estriol 3- and 16-glucuronides in pregnancy urine by column-switching high-performance liquid chromatography with fluorescence detection

An HPLC method for the direct and simultaneous determination of estriol 3- and 16-glucuronides in pregnancy urine is described. The method is based on direct derivatization of the glucuronic acid moiety in estriol glucuronides in urine with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution (or urine sample) in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at 37°C. The resulting fluorescent derivatives were separated by column-switching chromatography using a first column (YMC-Pack C₄) for clean-up of the derivatives and a second column (YMC Pack Ph) for the complete separation of the derivatives. The derivatives were detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise ratio=3) for estriol 3- and 16-glucuronides were 150 and 180 fmol in a 5 μl of urine (14 and 17 ng ml⁻¹ urine), respectively. The present method is highly sensitive and simple without any clean-up such as conventional solid-phase extraction.     

Bioluminescent enzyme immunoassay for estriol. Use of reversibly inactivated bacterial luciferase as label

A bioluminescent enzyme immunoassay using estriol labeled with reversibly inactivated bacterial luciferase is described. An estriol derivative bearing an alkylthiolsulfonate is linked to the cysteinyl thiols of luciferase by formation of mixed disulfide linkages; thus, luciferase becomes inactive. After immunoassay, the inactive luciferase of the label bound to the immunoprecipitate is reactivated by incubation with dithiothreitol and the luciferase activity then is quantitated by a 20-s reaction performed with an automated luminometer (LKB 1251). Under the defined conditions, the labels are stable for at least 14 days as tested at 4 degrees C. A standard curve with a wide linear range from 50 to 6000 pg is demonstrated. This unique technology discussed here, therefore, offers exciting possibilities as a sensitive and rapid enzyme immunoassay for estriol.     

Simultaneous determination of adreno-ovarian steroids from a single aliquot

A standardized technique for simultaneous fractionation and estimation of samples of progesterone, estrone, 17alpha-estradiol, 17beta-estradiol, estriol, corticosterone, cortisone, and cortisol obtained from guinea-pigs during mid-pregnancy is presented. The hormones were separated on a single TLC plate using a single solvent system, and were measured on a single biphasic column on the basis of a single aliquot. The technique affords considerable utility in the inves tigation of the complexities of the adreno-genital syndrome. Progesterone content in the ovaries and plasma showed an increase of 77% during pregnancy, while plasma levels of estrone were increased by 34%. Urinary estradiol levels were also markedly increased. Plasma estradiol and estriol and placental estriol could not be detected by the technique. Adrenal cortisone levels and plasma cortisone and cortisol were considerably higher during pregnancy. An initial overlap was found between cortisol and estriol on the TLC plate, though subsequent estimation by GLC overcome the problem.     

Synthesis of new steroid haptens for radioimmunoassay. Part IV. 3-O-carboxymethyl ether derivatives of estrogens. Specific antisera for radioimmunoassay of estrone,estradiol-17β, and estriol

The syntheses of 3-O-carboxymethyl ether derivatives of estrone, estradiol-17β, and estriol and the preparation of their bovine serum albumin (BSA) conjugates are described. These conjugates were employed for the generation of specific antisera suitable for radioimmunoassay (RIA) of estrone, estradiol-17β, and estriol. The previous concept that specific antisera for estrogens cannot be obtained by employing estrogens derivatized at the 3-position is unfounded.     

A simple radioimmunoassay for estriol 3-sulfate in pregnancy plasma without deconjugation

A highly specific anti-estriol 3-sulfate antiserum was treated with 50% ammonium sulfate, and the crude globulin fraction was coupled to CNBr-activated Sepharose-4B. Addition of 0.1M Tris-HC1 buffer (pH 8.3) containing 0.1M glutamine to the solution of antigen-antibody enabled assaying without solvent-extraction or chromatography to remove endogenous interference. Subsequently, a direct radioimmunoassay using [6,7-3H]-estriol 3-sulfate as a radioactive ligand without deconjugation has been established and applied to the determination of estriol 3-sulfate levels in pregnancy plasma. The increasing plasma levels of estriol 3-sulfate are correlated with estriol levels over the period of gestation. The mean values of sulfated estriol concentration (A) in late pregnancy plasma were approximately 7 times as high as unconjugated estriol (B), but individual ratios of A to B showed considerable variability.     

Production of monoclonal antibodies to estriol and their application in the development of a sensitive nonisotopic immunoassay

The development of highly specific monoclonal antibodies to estriol and a nonisotopic immunoassay (EIA) for unconjugated estriol based on the use of these monoclonal antibodies have been described. The monoclonal antibodies show little cross reactivity with other steroids and steroid conjugates and can be used directly in immunoassays without any purification. The EIA described here can be performed in 96-well microtiter plates or polystyrene tubes that have been coated with estriol-bovine serum albumin conjugate. In this assay, estriol in the standard or clinical samples (serum or saliva) competes with the immobilized steroid on the plate or the tube for binding with the antibody. The assay shows good agreement with radioimmunoassay (RIA) and is highly sensitive and reliable. Since no prior processing or extraction of the clinical samples is necessary, the method is potentially applicable for routine use in fetal monitoring as well as in a steroid laboratory.