Cortisol production rates in subjects with suspected Cushing's syndrome: assessment by stable isotope dilution methodology and comparison to other diagnostic methods.

It can be difficult to establish the diagnosis of Cushing's Syndrome (CS) in patients with mild or nonspecific clinical and biochemical findings, because available diagnostic tests have limited predictive values. We hypothesized that measurement of 24-h cortisol production rates (CPRs) might be a more sensitive indicator of CS in such patients. We measured CPRs in 28 patients with suspected CS (but equivocal biochemical findings) and in 22 healthy control subjects, by infusing tracer amounts of deuterated cortisol, with simultaneous measurements of 24-h urine free cortisol (UFC) levels; and we frequently sampled serum cortisol levels. CPRs were calculated from the ratio of isotopic enrichment to isotopic dilution of cortisol measured by gas chromatography-negative ion chemical ionization mass spectrometry. Nine of the patients proved to have CS by surgery (CS-Yes), whereas 19 patients were determined not to have CS by biochemical testing (CS-No). Mean 24-h UFCs, nocturnal serum cortisol levels, and CPRs were higher in CS-Yes, compared with CS-No and normal subjects. However, one CS-Yes patient had a normal 24-h UFC, two had normal nocturnal serum cortisol levels, and two had normal 24-h CPRs. There was extensive overlap in all of the biochemical parameters between the CS-Yes and the CS-No groups. Thus, measurement of CPR does not seem to offer any diagnostic advantage over available tests for the diagnosis of CS. Patients with proven CS can have normal UFC levels, normal CPRs, or normal nocturnal cortisol levels, whereas patients not thought to have CS may have elevated levels of any one or more these parameters.

Resistance to several steroids in two sisters.

A 14-yr-old native American girl from the Iroquois Nation was referred as a potential patient with the syndrome of apparent mineralocorticoid excess. Instead, her evaluation revealed resistance to glucocorticoids, mineralocorticoids, and androgens, but no resistance to vitamin D or thyroid hormones. She lacked Cushingoid features despite significantly high cortisol levels. Menstruation was regular, and there was no clinical evidence of masculinization despite high serum androgen levels in the male range. The patient's sister had similar clinical features. Partial resistance to exogenous glucocorticoid and mineralocorticoid administration was well demonstrated in both patients. It is proposed that these patients represent the first cases of partial resistance to multiple steroids, possibly due to a coactivator defect.

Cortisol production rate measurement by stable isotope dilution using gas chromatography-negative ion chemical ionization mass spectrometry.

Presented here is a stable isotope dilution technique for determining cortisol production rate (CPR). The method involves extraction and derivatization of cortisol isoforms from serum (0.5 ml), separation of derivatives by gas chromatography, and detection by using negative ion chemical ionization mass spectrometry. This method provides 50-100-fold greater sensitivity than positive ion mass spectrometry and allows for estimations of cortisol production rate with the use of small amounts of pooled serum, even in the presence of high concentrations of lipophilic contaminants. The area under the curve for the total selected ion chromatogram of fluoroacyl derivatives of cortisol (d0, m/z 782) and deuterated cortisol (d3, m/z 785) were used to determine the isotopic dilution ratio in three types of samples: 1) standards: d0/d3 ratios ranging from 1 to 8%; 2) controls: d3-cortisol added to serum with known cortisol concentration; 3) subjects: 24-h pooled serum samples (q 30 min over 24 h) from healthy children (male 10-13 years; female 7-11 years) receiving continuous infusions of d3-cortisol at 2-4% of their estimated CPR. Recovery after the solid phase extraction and derivatization process was >90%, as determined by thin-layer chromatography. Expected versus measured ratios for d3/d0 in standards and serum controls were highly correlated (r2(standard) = 0.99; r2(control) = 0.99) over a wide range of d3-cortisol enrichment (1.0-10.0%). Mean 24-h CPRs were 4.8 +/- 0.6 mg/m2/24 h (mean +/- SEM, n = 7) in male children and 4.4 +/- 0.5 mg/m2/24 h in female children (n = 4). These CPR values are lower than those derived by radio tracer methods, but are in agreement with previous isotopic dilution studies. This technique is an important tool for assessing CPRs in a wide range of disease states affecting cortisol production.

Rapid loss of oestrogen and progesterone receptors in human leiomyoma and myometrial explant cultures.

Oestrogen and progesterone are promoters of uterine leiomyoma growth: oestrogen receptors (ER) and progesterone receptors (PR) are over-expressed in these tumours. Paradoxically, there is a heterogeneity in responsiveness of leiomyoma growth to oestrogen and progesterone in culture. In this study, leiomyoma and adjacent myometrium were obtained at hysterectomy. The effect of oestrogen and progesterone on steroid receptor maintenance was examined using minced explants. Quantitative enzyme-linked immunoassay and Northern analysis were performed to assess ER and PR protein and mRNA content respectively. There was an approximately 75% decrease in ER and PR protein content within 8 h of incubation in both leiomyoma and myometrium. The presence or absence of oestrogen and/or progesterone had no effect on receptor protein loss. Northern analysis indicated a parallel loss of ER and PR mRNA transcripts. These findings suggest that the ER and PR expression in leiomyoma may require other extracellular factors. In-vitro studies designed to test the effects of sex steroids and their respective inhibitors on growth and function of leiomyoma and myometrial cells should consider this phenomenon.

Estrogen receptor gene expression in human uterine leiomyomata.

Estrogen and progestin are believed to be important physiological regulators of uterine leiomyoma growth. We recently showed that progesterone receptor messenger ribonucleic acid (mRNA) and protein levels are increased in human uterine leiomyomas compared with those in myometrial biopsy tissue obtained from the same patient. To further characterize the molecular mechanisms underlying abnormal growth of uterine leiomyomas, we analyzed biopsy samples of tumor and adjacent normal myometrium for estrogen receptor (ER) gene expression. Northern analysis indicated that ER mRNA levels were increased 1.4-to 12.6-fold in leiomyoma compared with myometrium in all patients examined (n = 11), whereas beta-actin mRNA was not different between the two groups. The size of the primary ER mRNA transcript was 6.2 kilobases in both leiomyoma and myometrium, indicating no gross mutation of the ER gene. An ER protein of 66 kilodaltons was detected by Western blot analysis, and quantitative immunoassay of ER revealed 9448 +/- 1955 fmol/mg DNA in leiomyoma compared to 2827 +/- 979 fmol/mg DNA in myometrial tissue. Scatchard analysis of 17 beta-estradiol binding to cell-free extracts revealed enhanced binding capacity (per mg DNA) in leiomyoma tissue (n = 6) of about 6-fold, whereas ER binding affinity was not substantially different between the leiomyoma and adjacent myometrial tissues. We propose that increased expression of progesterone receptor in leiomyoma is most likely a consequence of overexpression of functional ER that results in increased end-organ sensitivity to estradiol.

Progesterone receptor messenger ribonucleic acid and protein are overexpressed in human uterine leiomyomas.

OBJECTIVE: Our purpose was to identify molecular mechanisms underlying abnormal growth of uterine leiomyomas. STUDY DESIGN: Biopsy samples of tumor and adjacent "normal" myometrium from nine patients were analyzed for progesterone receptor gene expression and for proliferation-associated antigen Ki-67. RESULTS: Northern analysis indicated that progesterone receptor messenger ribonucleic acid levels were increased twofold to 15-fold in leiomyoma compared with adjacent myometrial biopsy tissue from all patients (n = 9), whereas beta-actin messenger ribonucleic acid was at similar levels in these samples. Quantitative immunoassay, immunohistochemistry studies, and Western blot analyses revealed increased amounts of progesterone receptor protein in the tumor tissue. Both the progesterone receptor A and B forms were expressed in the leiomyoma and adjacent myometrium. Corresponding to increased progesterone receptor gene expression, the proliferation-associated antigen Ki-67 was also significantly elevated in the leiomyoma tissue. CONCLUSION: These data provide the first evidence that progesterone receptor messenger ribonucleic acid is overexpressed in uterine leiomyomas, suggesting that amplified progesterone-mediated signaling is instrumental in the abnormal growth of these tumors.

Glucocorticoids inhibit estradiol-mediated uterine growth: possible role of the uterine estradiol receptor.

Stress-related activation of the hypothalamic-pituitary-adrenal axis (HPA) is associated with suppression of the reproductive axis. This effect has been explained by findings indicating that corticotropin-releasing hormone suppresses hypothalamic gonadotropin-releasing hormone (GnRH) secretion via an opioid peptide-mediated mechanism, and that glucocorticoids suppress both GnRH and gonadotropin secretion and inhibit testosterone and estradiol production by the testis and ovary, respectively. To evaluate whether glucocorticoids suppress the effects of estradiol on its target tissues, we examined the ability of dexamethasone to inhibit estradiol-stimulated uterine and thymic growth in ovariectomized rats. Estradiol alone, given daily for 5 days, caused dose-dependent uterine and thymic growth. Dexamethasone alone, given daily for 5 days, caused a dose-dependent decrease in body weight gain and in thymic growth. When estradiol and dexamethasone were administered simultaneously, however, body weight gain and thymic growth were also inhibited (p less than 0.05). Dexamethasone decreased estradiol-induced uterine cytosolic and nuclear estrogen receptor concentrations (E2 R0, p less than 0.05; E2nR0, respectively), but had no effect on estradiol-induced progesterone receptor concentrations (P4R0, p greater than 0.05). Levels of uterine glucocorticoid receptors were not affected by estrogen and/or dexamethasone treatment. These findings suggest that stress levels of glucocorticoids, administered over a 5-day interval, block the estradiol-stimulated growth of female sex hormone target tissues. This effect may be partially mediated by a glucocorticoid-induced decrease of the estradiol receptor concentration. Thus, another mechanism by which the HPA may influence reproductive function during stress is by a direct effect of glucocorticoids on the target tissues of sex steroids.

Glucocorticoid resistance in humans and nonhuman primates.

In humans, the syndrome of cortisol resistance is characterized by the absence of signs and symptoms of Cushing's syndrome, elevated total and unbound plasma cortisol concentrations, and increases in urinary free cortisol excretion and plasma adrenocorticotropic hormone. In one family, a severely affected member had hypertension and hypokalemic alkalosis associated with increased plasma concentrations of corticosterone and deoxycorticosterone. These patients are resistant to suppression of the pituitary-adrenal axis by dexamethasone. Dexamethasone therapy, however, effectively corrected hypertension and hypokalemic alkalosis in the severely affected patient, without causing signs of glucocorticoid excess. The glucocorticoid receptor from these patients has a low affinity for glucocorticoids and is unstable during thermal activation. Both the molecular weight of the glucocorticoid receptor and the size of the corresponding mRNA are similar to those of normal controls. Transformation of B-lymphocytes with Epstein-Barr virus leads to induction of glucocorticoid receptors. Receptor induction, however, is lower in patient cells than those obtained from normal controls. This decreased induction parallels decreased expression of glucocorticoid receptor mRNA. Thus, in this form of glucocorticoid resistance the glucocorticoid receptor is abnormal and leads to diminished target organ responsiveness. Many New World primates exhibit glucocorticoid "resistance," without apparent pathology. These species have markedly elevated plasma cortisol, both total and unbound concentrations, increased urinary free cortisol excretion, and marked increases in plasma adrenocorticotropic hormone and beta-endorphin. The glucocorticoid receptors of these primates have decreased affinity for glucocorticoids, are thermolabile, and are not induced by Epstein-Barr virus transformation as indicated by specific binding and mRNA expression. Both the molecular weight of the glucocorticoid receptor and the size of the corresponding mRNA are similar to those of normal controls. Despite the high plasma cortisol concentrations in these primates, there is no sodium retention and aldosterone levels are actually increased. The kidney aldosterone receptor cross-reacts poorly with cortisol, explaining the absence of sodium retention. New World primates also have progesterone, estrogen, aldosterone, and vitamin D insensitivity, suggesting a common factor linking steroid hormone receptors.

Glucocorticoid receptor-mediated effects on rat fibrosarcoma growth.

Glucocorticoid receptors are present in most normal and malignant mammalian cells. To examine the hypothesis that the growth of methylcholanthrene-induced malignant sarcoma is glucocorticoid dependent, we evaluated the behavior of malignant fibrosarcoma (MCA) in adrenalectomized rats treated with either normal saline or deoxycorticosterone acetate and in intact rats treated with placebo or with the glucocorticoid receptor antagonist RU 486. Survival, tumor weight, and loss of body weight (an index of cachexia) were measured. In MCA-bearing rats, neither survival nor loss of body weight was affected by bilateral adrenalectomy or by treatment with RU 486. Tumor weight and time-integrated tumor volume, however, were significantly less in bilaterally adrenalectomized rats without deoxycorticosterone acetate replacement than in animals treated with deoxycorticosterone acetate. Similarly, tumor weight and time-integrated tumor volume were less in intact animals treated with RU 486 than in intact animals treated with placebo. The glucocorticoid receptors in the tumor cells had similar binding capacity (Ro) and equilibrium dissociation constant (Kd) as in control rat fibroblasts. These results suggest that the growth of MCA sarcoma cells is partially dependent upon glucocorticoids. This effect of glucocorticoids, however, was not of sufficient magnitude to improve survival and prevent cachexia. We conclude that glucocorticoids appear to influence MCA sarcoma growth in the rat, and that glucocorticoid receptor blockade, perhaps in combination with other antitumor agents, merits future study in the treatment of malignant tumors.

Pharmacokinetic properties of the antiglucocorticoid and antiprogesterone steroid RU 486 in man.

RU 486 (17 beta-hydroxy-11 beta-[4-dimethylamino phenyl]-17 alpha-[1-propynyl]estra-4,9-dien-3-one) is a clinically useful glucocorticoid and progesterone antagonist. The authors studied the pharmacokinetic properties of this drug in normal volunteers and patients with Cushing's syndrome using a rat progesterone radioreceptor assay. This assay gave values similar to those obtained with a rat glucocorticoid radioreceptor assay. After a single oral dose of 25 mg/kg (n = 11) or 10 mg/kg (n = 11) to normal volunteers, plasma concentrations of progesterone receptor-reactive material reached maximal levels of 754 +/- 288 (mean +/- S.D.) and 517 +/- 183 micrograms/dl. This occurred at 3.1 +/- 1.9 and 2.5 +/- 1.0 h, respectively. The respective apparent plasma half-lives were 19.2 +/- 7.0 and 20.6 +/- 7.7 h. Four patients with Cushing's syndrome treated chronically (10-20 mg/kg/day) had relatively constant plasma levels of receptor reactivity ranging from 506 to 1184 micrograms/dl. Chromatographic characterization of circulating receptor reactivity showed that the active fraction corresponded to RU 486 and its hydrophilic N-mono- and N-didemethylated metabolites. Less than 0.5% of the daily dose was excreted in the urine of two of these patients as receptor reactivity. The drug bound extensively to circulating albumin, which competed with the glucocorticoid receptor of intact human mononuclear leukocytes for [3H]RU 486 in a concentration-dependent manner.

Glucocorticoid receptors in Epstein-Barr virus-transformed lymphocytes from patients with glucocorticoid resistance and a glucocorticoid-resistant New World primate species.

Members of a previously reported family with glucocorticoid resistance and several New World primates have high plasma cortisol concentrations without any signs of glucocorticoid excess. The glucocorticoid receptor in circulating leukocytes and cultured skin fibroblasts from these patients and the animals is characterized by a decreased affinity for dexamethasone. On the other hand, the cell content of receptor is similar to that of corresponding tissues of normal humans. Detailed biochemical-biophysical studies of the glucocorticoid receptor in this familial syndrome and animal model became possible with the use of Epstein-Barr virus-transformed lymphocyte lines. Cell lines from patients with this syndrome and from the marmoset (Saguinus oedipus) contained decreased amounts of glucocorticoid receptors with concomitant decreases in nuclear receptor content compared to cultured Epstein-Barr virus-transformed lymphocytes from normal human subjects. This may reflect diminished induction of glucocorticoid receptor during viral transformation of cells from the patients and the animal model. Receptors from a severely affected glucocorticoid-resistant patient and the marmoset had decreased affinity for dexamethasone. Evidence for a mild affinity defect of the glucocorticoid receptor in a patient with asymptomatic glucocorticoid resistance was obtained by increased hormone-receptor dissociation at an elevated temperature. Thermal stability, mero-receptor formation, thermal activation of cytosolic receptor, and mol wt of receptors from all cell lines were normal. Only the receptors of the severely affected patient had a discernible defect in temperature-induced activation of intact cells. We conclude that the major detectable change in the receptor in both the patients and the animal model is the decreased affinity for glucocorticoid. Viral receptor induction is decreased in both patient and marmoset cells. The physiological relevance of this phenomenon is not known. Gross receptor molecule changes or changes in its stability at higher temperatures were not found. Mixing studies did not show involvement of cytosolic modifiers or inhibitors. Mutation(s) of the receptor molecule leading to low affinity for the hormone is the most likely explanation of the isolated glucocorticoid resistance in the patients. The glucocorticoid resistance of the New World primate, which is part of generalized steroid hormone resistance, appears to be a result of more complex changes.

The new world primates as animal models of glucocorticoid resistance.

Many New World primate species have greatly increased plasma cortisol concentrations, decreased plasma cortisol binding globulin capacity and affinity, marked resistance of the hypothalamic-pituitary-adrenal axis to suppression by dexamethasone, and no biological evidence of glucocorticoid excess. These primates also have high levels of circulating progesterone, estrogen, mineralocorticoid, androgen and vitamin D. The glucocorticoid target tissues that have been examined (circulating mononuclear lymphocytes and cultured skin fibroblasts) have normal concentrations of glucocorticoid receptors with decreased affinity for dexamethasone. Transformation of B-lymphocytes with the Epstein-Barr virus leads to glucocorticoid receptor induction that is less than that observed with cells from Old World primates. The receptor in these cells has a low affinity for dexamethasone. The low affinity leads to an increased loss of specific bound ligand during thermal activation. Meroreceptor generation is normal. The molecular weight of the receptor, determined by SDS-PAGE, is similar to that of Old World primates (approximately 92,000) and the activation pattern per se, examined in vitro by heating cytosol and performing phosphocellulose chromatography, appears similar to that of human controls. The ratios of nuclear to cytosolic hormone-receptor-complexes and of cytosolic activated to unactivated receptor complexes in intact cells are similar to Old World primates. Results from mixing studies do not support the hypothesis that a binding inhibitor(s) or a deficient cytosolic positive modifier(s) of binding underlies the findings in these primates. The New World primates, unlike men with the syndrome of primary cortisol resistance, have compensated for their condition with intra-adrenal and mineralocorticoid receptor adaptations. Thus, unlike Old World primates, cortisol in New World primates has only weak sodium-retaining potency because the aldosterone receptor has a low affinity for cortisol. The common element that would explain the apparent resistance to six steroid hormones in New World primates remains unknown.

Cortisol resistance in man.

Primary cortisol resistance in man is a familial disease characterized by increased plasma cortisol concentrations, high urinary free cortisol excretion, a normal circadian pattern of cortisol secretion, resistance to adrenal suppression by dexamethasone and absence of the clinical stigmata of Cushing's syndrome or signs of adrenal insufficiency. In its severe form, hypertension and hypokalemic alkalosis are present, owing to increased secretion of the sodium-retaining corticoids, corticosterone and deoxycorticosterone. In subjects with a less severe resistance to cortisol, there are no clinical abnormalities and the disease is revealed only by detailed examination of several parameters of cortisol metabolism or by glucocorticoid receptor studies. In whole-cell glucocorticoid receptor assays (peripheral mononuclear leukocytes, fibroblasts, or B-lymphocytes transformed with the Epstein-Barr Virus) low receptor affinity for dexamethasone could be demonstrated conclusively only in the severely affected subject. When affected cells are transformed with the Epstein-Barr virus, receptor induction is less than that of normal cells. The decreased affinity of the receptor for its ligand is reflected in an increased rate of loss of specific bound ligand during thermal activation. The molecular weight of the receptor, determined by SDS-PAGE, is similar to that from normal cells (approximately 92,000). Only in the severely affected patient was the proportion of activated receptor remaining in the cytosol of thermally activated intact cells reduced. At saturating concentrations of dexamethasone, nuclear binding appears normal in cells from both the severe and the asymptomatic forms of this condition, providing an explanation for the apparently complete compensation of the target tissue resistance to glucocorticoids by the high plasma cortisol levels. The clinical manifestations of the disorder (hypertension, hypokalemia) can be corrected with high doses of dexamethasone (3mg/day).

Glucocorticoid receptors in Epstein-Barr virus-transformed human lymphocytes.

Glucocorticoid receptors were studied in cultured human lymphocytes from normal donors after transformation with the Epstein-Barr Virus (EBV) and compared to those of circulating human mononuclear leukocytes. Both whole cell and cytosol fractions were examined for [3H]-dexamethasone binding. The concentration and absolute number of glucocorticoid binding sites were increased five-fold in the transformed cells when compared to the non-transformed human mononuclear leukocytes. However, the affinity (Kd) of the glucocorticoid receptor for dexamethasone was the same in both types of cells. The denatured glucocorticoid receptor, covalently labelled with [3H]-dexamethasone-21-mesylate, was identified by SDS polyacrylamide gel electrophoresis as a protein moiety with Mr approximately equal to 92,000, similar to that obtained from human non-transformed mononuclear leukocytes. The pattern of the activation of the hormone-receptor complexes, analyzed by phosphocellulose chromatography, was similar in both types of cells, and also the time-courses of loss of specific binding during thermal activation were similar. These results suggest that viral transformation is associated with increases in the concentration and the absolute number of glucocorticoid receptors whereas other qualitative receptor characteristics remain similar. Thus, transformation of cells with EB virus can provide a constant source of glucocorticoid receptors for study.

Radioimmunoassay and metabolic clearance rate of catecholestrogens, 2-hydroxyestrone and 2-hydroxyestradiol in man.

Plasma levels of 2-hydroxyestrone (2-OHE1) and 2-hydroxyestradiol (2-OHE2) were determined by a new radioimmunoassay which employed a short Sephadex LH-20 column chromatography for the purification of samples and the antiserum to 2-hydroxyestrone-17-(O-carboxymethyl)oxime-BSA conjugate for assay. The plasma value was below the detection limit for the assay (approximately 15 pg/ml) in men and non-pregnant women, but rose 20-200 pg/ml during pregnancy in 2-OHE1 and around 15 pg/ml in the 3rd trimester of pregnancy in 2-OHE2. There was no significant difference of plasma 2-OHE1 level between normal pregnancy and toxemic pregnancy with hypertension, in the 3rd trimester. The plasma level was very low in all of three subjects with the placental dysfunction in toxemic pregnancy. The plasma metabolic clearance rate (MRCp) of 2-OHE1 and 2-OHE2 were determined in normal adults by two methods; infusion of unlabeled 2-OHE1 and 2-OHE2 to equilibrium with radioimmunoassay of their plasma levels, and infusion of [3H]-2-OHE1 and [3H]-2-OHE2 to equilibrium with measurement of chromatographically purified their tritium. The MCRs by the former and latter methods was 40-70 X 10(3) and 15-50 X 10(3) in 2-OHE1, and 18-29 X 10(3) and 12-14 X 10(3) l/day in 2-OHE2, respectively. The major plasma metabolite comigrated with 2-methoxy compounds to each catecholestrogen. The t1/2 of disappearance rate by the method of infusion of unlabeled compounds was approx. 45 s in 2-OHE1 and 90 s in 2-OHE2. When [3H]-2-OHE1 and [3H]-2-OHE2 were incubated with blood samples of adults, 2-methoxy compounds also rapidly formed. From these results it is concluded that the extremely high MCRp of 2-OHE1 and 2-OHE2 make it unlikely these compounds circulate peripherally except in pregnancy in levels sufficient to produce the physiological effects on estrogen receptors or catecholamines.

Radioimmunoassay and metabolism of the catechol estrogen 2-hydroxyestradiol.

Plasma levels of 2-hydroxyestradiol (2-OHE2) were measured using a new RIA procedure. Values were below the detection limit of the assay (less than 10 pg/ml), except in the third trimester of pregnancy, when they rose to approximately 15 pg/ml. The infusion of 130 microgram/h purified 2-OHE2 elevated its plasma concentration to 155 pg/ml, consistent with a plasma MCR (MCRp) of approximately 20,000 liters/day. The infusion of [3H] 2-OHE2 to equilibrium and chromatographic separation of the extracted plasma metabolites yielded an MCRp of about 13,000 liters/day; the major plasma metabolite comigrated with 2-methoxyestradiol, and [3H] xi-methoxyestrone was also formed. The MCRp, of 2-OHE2 is approximately half that of 2-hydroxyestrone (2-OHE1), but much higher than those of other steroids. As is true for 2-OHE1, the clearance of 2-OHE2 must occur primarily in the blood compartment. Together, the measured MCRp values and estrogen receptor affinities of 2-OHE2 and 2-OHE1 predict a relative potency for effects upon gonadotropin secretion which is close to that observed in vivo.

Metabolic clearance rate and uterotropic activity of 2-hydroxyestrone in rats.

2-Hydroxyestrone (2-OHE1) has much lower uterotropic potency than might be predicted from its uterine estrogen receptor affinity. 2-OHE1 displaces saturably bound [3H]estradiol from rat uterine cytosol with a competitive inhibition constant of 8.6 nM, while the dissociation constant for 17 beta-estradiol (E2) is 0.42 nM. From this ratio of binding affinities, one would expect some agonist or antagonist activity of 2-OHE1 to be apparent at doses roughly 20-50 times the minimum effective dose of E2. Instead, at doses of 2-OHE1 1000 times an effective dose of E2, no uterotropic effect was observed. When 2-OHE1 was injected together with E2 at dose ratios of 500:1, there was no antagonism of the effect of E2. To examine this discrepancy, the plasma MCRs (MCRpS) of E2 and 2-OHE1 were determined by continuous infusion techniques. Plasma concentrations of 2-OHE1 and E2 during control and infusion periods were measured by RIAs. The MCRp of 2-OHE1 averaged 50,000 ml/h, more than 100 times that of E2 (approximately 400 ml/h). The extraordinarily high MCRp of 2-OHE1 may explain the failure to observe any biological effects of this catechol estrogen, even at high doses. This rapid metabolism, presumably occurring in the blood compartment, should be considered in handling blood samples for RIA and in devising studies of the actions of catechol estrogens.