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Depression has been commonly associated with type 1 diabetes (T1D) and insulin covalently modified with catecholestrogens (CEs) was found in serum of these T1D patients. This study aimed to know whether depression link to higher antibodies against estrogenized insulin in T1D. ELISA (direct binding and competition) and quantitative precipitin titration were used to detect antibodies and their affinities against estrogenized insulin in the serum of 66 depressed T1D (DT1D) patients (out of 110 T1D) and 41 control subjects. Antibodies from DT1D patients showed high binding specificity to estrogenized insulin (2-hydroestradiol-insulin; 2-OHE 2 -Ins) in comparison to overall T1D patients (p < 0.05) or control subjects (p < 0.001). However, T1D sera demonstrate high recognition to 2-OHE 2 -Ins as compared to Ins (p < 0.05) or 2-OHE 2 (p < 0.001). The affinity of antibodies from DT1D and T1D patients was 1.32 × 10 -7 M and 1.43 × 10 -7 M, respectively. Depression linked to higher antibodies production against estrogenized insulin in T1D. Furthermore, depression in T1D generates inflammatory conditions that further increased antibodies production in T1D patients., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The interaction of estrogens with environmental toxins in free radicals generation: reactive oxygen species (ROS) or reactive nitrogen species (RNS) which participates in cancerogenesis is not yet recognized. Chromium(VI) is widely present in environment. One of its toxicity pathway is free radicals generation. Estrogens have the ability to scavenge free radicals, but may also act as prooxidants. Both chromium(VI) and estrogens are classified by International Agency for Research on Cancer (IARC) as carcinogens, so synergistic effect seems very dangerous. The interaction of chromium and estrogens in ROS generation are partly described but there are no reports on estrogen/chromium interaction on nitric oxide (NO) generation. The aim of the study was to examine the interaction of chromium(VI) and 17-p-estradiol (E2) on NO level in human blood as well as the role of E2 metabolites: 4-hydroxyestradiol (4-OHE2) and 16a-hydroxyestrone (16α-OHE1) in these processes. The NO level was estimated with the diagnostic kit (Nitric Oxide Colorimetric Detection Kit from Arbor Assays) in human blood in vitm. The results showed that Cr(VI) in used concentration (0.5; 1.0 and 5.0 gg/mL) decreases significantly NO level in blood, acting antagonistically to E2 and 4-OHE2. Estrogens (E2, 4-OHE2 and 16α-OHEI) do not protect against inhibiting effect of Cr(VI) on nitric oxide generation in blood because after combined exposure the decreased production of NO in blood was noted. In conclusion, presented results provide the information about the character of estrogen/Cr(VI) interaction in NO level in human blood. It is important knowledge for cardio protected effect e.g., hormone replacement therapy in environmental or occupational exposure to Cr(VI), chromium supplementation, also important for cancer risk evaluation.

Protein adducts covalently modified by catechol estrogens (CEs), referred to as estrogenized proteins, are potential biomarkers for estrogen homeostasis or exposure to environmental toxicants. However, serum proteins endogenously modified by CEs and the modification sites remain elusive. In this study, liquid chromatography-mass spectrometry (LC-MS)-based shotgun proteomics is applied to identify site-specific protein estrogenization in human blood via a systematic approach and stringent validation. We showed CEs, namely 2- and 4-hydroxyl estrogens which are regarded as biomarkers for estrogen homeostasis, form covalent bonds with proteins, mainly via side chain Cys, Lys, or His residue. Estrogenization of purified human serum albumin (HSA) and immunoglobulin G (IgG) at specific sites was achieved by co-incubation and used as the standards to confirm the identified estrogenization in serum proteins. Based on a database search, estrogenized peptides derived from serum proteins in patient blood were identified; endogenous estrogenization of HSA and IgG-1 at multiple sites were confirmed as compared to the standards. Based on a test using Ellman's reagent, estrogenization produced stable products and irreversibly abolished the reactivity of Cys34-HSA, which is the most important antioxidant and nitric oxide carrier in blood. Given the importance of estrogen metabolism in environmental toxicology, further exploration of estrogenized proteins is warranted for biomarker discovery and/or new mechanisms in disease process., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The origin of human tumors has been attributed to the exposure to several environmental chemicals and implicated in the increase of incidence in breast cancer. Progression of breast cancer follows a complex multistep process that seems to depend on various exogenous and endogenous factors. The aim of this study was to examine the effects of the organo-phosphorous pesticide malathion in the presence of estrogen on neoplastic transformation of rat mammary glands. Virgin female rats were sacrificed after 30, 124 and 240 days of 5-day injections twice a day. There were four groups: i) control, ii) malathion (22 mg/100 g body weight, BW), iii) 17β-estradiol (30 µg/100 g BW) and iv) combination of both. Progressive alterations in ducts were observed by the effect of malathion in comparison to control after 240 days. Ducts markedly increased in size and number of cells per square millimeter and tumors similar to ductal carcinoma were originated. The increase in number of proliferative ducts per square millimeter was significantly (P<0.05) higher in malathion-treated animals compared to the other groups. Progressive alterations in lobules with estrogen treatment were found after 240 days. Lobules became markedly abnormal, referred to as secretory lobules, increased in number and size and the tumors originated were similar to lobular carcinoma. The increase in number of secretory lobules was significantly (P<0.05) higher in estrogen- treated animals compared to the other groups. Treatment with the combination of malathion and estrogen gave rise to tumors constituted of both proliferative ducts and secretory lobules as well as formation of estrogen metabolites such as 2 and 4 catechol estrogens in the blood of the animals after 240 days. We concluded that morphological changes and alterations in the blood of the animals can be used as biomarkers for mammary gland cancer.

The liquid chromatography (LC) with electrochemical detection allows to determine, evaluate and validate the level of estrogens and their metabolites in serum. The method is fast and sensitive, and the hormones can be determined simultaneously, from one serum sample. The proposed method was successfully applied to the determination of following estrogens: estrone (E(1)), 17beta-estradiol (E(2)), estriol (E(3)) and following catecholestrogens: 2-hydroxyestradiol (2-OHE(2)), 4-hydroxyestradiol (4-OHE(2)), 4-hydroxyestrone (4-OHE(1)), 2-methoxyestradiol (2-MOE(2)) and 2-methoxyestrone (2-MOE(1)). The method of LC with electrochemical detection (LC/EC) was applied for the determination of catecholestrogens in serum sample taken from pregnant women. Estrogens and catecholestrogens were extracted from 1 mL of serum by applying diethyl ether under the specified conditions. The parameters of the procedure included using a specific mobile phase, applying the column Symmetry C18 (5 microm, 3.0 mm x 150 mm), equipping the applied electrochemical detector with the working glassy-carbon electrode, as well as applying the reference electrode Ag/AgCl. The calibration studies on this study were performed, and a good analytical performance for E(1), E(2), E(3), 2-OHE(2), 4-OHE(2), 4-OHE(1), 2-MOE(2) and 2-MOE(1) was attained, along with low limits of detection (LOD of 0.18-0.30 ng/mL), satisfactory limit of quantification (LOQ of 0.23-0.92 ng/mL) and excellent linear dynamic range (0.6-8.0 ng/mL). In conclusion, the presented methodology is the sensitive method of the simultaneous measurement of eight estrogens and their metabolites from one sample of blood and it might be clinically applied for testing serum of pregnant women, as well as for further studies on estrogens and their metabolites., ((c) 2009 Elsevier B.V. All rights reserved.)

A modified ELISA assay for measurement of the two estrogen metabolites 2-hydroxyestrone (2OHE1) and 16alpha-hydroxyestrone (16alphaOHE1) in plasma and serum has been developed. Previously, these have only been measured in urine. It is not known how well the measurements of these metabolites in urine and plasma are correlated. The goal of this study was to compare urinary and plasma levels of 2OHE1 and 16alphaOHE1 and their ratios and to explore how they were affected by ethnicity, dietary and genetic factors, and medication use. Blood and urine samples were obtained from 511 nulliparous women, aged 17-35, from four ethnic groups during the same visit at the study center, on a random day of the menstrual cycle. The overall correlation between the 2OHE1/16alphaOHE1 ratio in plasma and urine was fair (rs = 0.52; p < 0.0001). In general, the correlation between the 2OHE1/16alphaOHE1 ratio in urine and plasma was higher among women not using oral contraceptives (OCs) (rs = 0.58; p < 0.0001) than among women currently using OCs (rs = 0.34; p < 0.0001). The correlation was highest for samples obtained during the mid-cycle in among non-OC users (rs = 0.83; p < 0.0001). Among non-OC users, the urinary 2OHE1/16alphaOHE1 ratio was stable over the menstrual cycle while there was an increase in the plasma 2OHE1/16alphaOHE1 ratio. The strongest factors predicting discordance between the urinary and plasma 2OHE1/16alphaOHE1 ratios among non-OC users were a baseline urinary 2OHE1/16alphaOHE1 ratio in the three upper quartiles (p < 0.001), the menstrual cycle phase (p = 0.001), and the number of cups of coffee consumed per day (p = 0.006). Among current OC users, the strongest predictors of discordance between the urinary and plasma 2OHE1/16alphaOHE1 ratios were a baseline urinary 2OHE1/16alphaOHE1 ratio in the three lower quartiles (p < 0.001), being black (p = 0.001), and being Asian (p = 0.014). In conclusion, we found that the correlation between the two methods was fair and varied according to the baseline urinary 2OHE1/16alphaOHE1 ratio, ethnic group, OC status, coffee consumption, and time of menstrual cycle when the samples were obtained.

We have developed a gas chromatography/mass spectrometry (GC/MS) assay to measure 17beta-estradiol (E) and its biologically active metabolites 2-hydroxyestradiol (2OHE) and 4-hydroxyestradiol (4OHE), and 2-methoxyestradiol (2MEOE) and 4-methoxyestradiol (4MEOE) in rat plasma. All analytes are well separated and show a linear relationship between concentration (0.25-5 pg/microl) and signal, and coefficients of variation (CVs) are low. Intra-assay CV for the lowest quality control samples (QCs) (0.375 pg/microl) were on average for 17beta-estradiol 20.5%, for 2-hydroxyestradiol 15.6%, for 4-hydroxyestradiol 16.5%, for 2-methoxyestradiol 16.5%, and for 4-methoxyestradiol 11.5%. The inter-assay CVs for the lowest QCs were for 17beta-estradiol 12.1%, for 2-hydroxyestradiol 7.1%, for 4-hydroxyestradiol 15.5%, for 2-methoxyestradiol 16.7%, and for 4-methoxyestradiol 9.7%. The highest sensitivity for this assay was observed for hydroxyestradiols followed by the methoxyestradiols and 17beta-estradiol. In summary, we describe a convenient, sensitive, and specific assay to measure 17beta-estradiol and its biologically active metabolites.

2-Hydroxyestradiol 17-sulphate (2-OH E2-17-S) is a catecholized form of sulphated estrogen. In vitro studies showed that its antioxidative effect is almost equal to that of free catecholestrogens, such as 2-OH E2 or 4-OH E2 and alpha-tocoferol, but the existance of 2-OH E2-17-S in human serum has not yet been made clear. 2-OH E2-17-S strongly antagonizes lipid peroxidation, and so it may play an important role in pregnancy, for example as an anti-oxidant in pregnancy-induced hypertension (PIH). The serum level of 2-OH E2-17-S was measured during mid to late pregnancy by a direct radioimmunoassay (RIA) without hydrolysis. The serum levels at 28-31 weeks, 32-35 weeks and 36-40 weeks of gestation were 4.68+/-0.93 (mean+/-SE), 8.38+/-1.21 and 18.31+/-3.41 nmol/l, respectively. The serum level in PIH cases at 36-40 weeks (4.64+/-1.29 nmol/l) was significantly lower than that in normal pregnancy. The 2-OH E2-17-S level in umbilical arteries was significantly higher than that in maternal peripheral vein. These results suggest that the feto-placental unit plays an important role in catecholizing E2-17-S to 2-OH E2-17-S, which may act as an antioxidant in pregnancy.

The effects of 17 beta-estradiol and the important estrogen metabolites, 2-hydroxyestrone (2-OHE1) and 16 alpha-hydroxyestrone (16 alpha-OHE1) on bone, mammary gland, and uterine histology, and on blood cholesterol were investigated in ovariectomized growing rats. Rats were treated with 200 micrograms/kg of body weight/day of each of the test compounds for 3 weeks. Ovariectomy resulted in uterine and mammary gland atrophy, increased body weight, bone turnover and tibia growth, and hypercholesterolemia. 17 beta-estradiol treatment prevented these changes, with the exception that this high dose of estrogen did not prevent hypercholesterolemia. 2-OHE1 had no effect on any of the measurements. 16 alpha-OHE1 resulted in bone measurements that did not differ from the 17 beta-estradiol-treated rats and prevented the increase in serum cholesterol. In contrast, 16 alpha-OHE1 resulted in increases in uterine weight, uterine epithelial cell height, and mammary gland cell proliferation that were significantly less than the 17 beta-estradiol treatment. These findings demonstrate that 16 alpha-hydroxylation of estrone results in tissue-selective estrogen agonistic activity, whereas 2-hydroxylation resulted in no measured activity. Furthermore, they suggest that factors that modulate the synthesis of these metabolites could selectively influence estrogen target tissues.

A single-subject experimental design was used to obtain some preliminary findings on the plasma responses of catecholestrogens (CE) to acute exercise and brief, but exhaustive training on a cycle ergometer. One previously untrained eumenorrheic female (body fat: 26% VO2max: 43.3 ml x kg(-1) x min[-1]) participated in this study. Resting CE levels were for "total" (unconjugated + conjugated) 2-hydroxyestrogens (2-OHE) 162pg/ ml and 350 pg/ml in the follicular (FPh) and luteal phase (LPh), respectively. Plasma total 4-hydroxyestrogen (4-OHE) levels were 41 pg/ml in the FPh and 66 pg/ml in the LPh. For "total" 2-methoxyestrogens (2-MeOE), we found 257 pgl/ml in the FPh and 374 pg/ml in the LPh. Resting levels of 2-hydroxy CE following a period of brief, intensive training were decreased during the LPh (2-OHE: -38%; 2-MeOE: -19%), whereas 4-hydroxy CE were unaffected. After training, the formation of CE as expressed by the 2-OHE:E and 4-OHE:E ratios, was increased by 75% and 200% at rest, respectively. CE activity or O-methylation, as estimated from the 2-MeOE:2-OHE ratio, was higher following training (FPh: +22%; LPh: +30%). During acute exercise before training, we observed a small rise proportional to the exercise intensity in the plasma "total" primary estrogen concentrations (FPh: +28%; LPh: +16%), and no changes in either 2-OHE or 2 MeOE levels. Plasma concentrations of 4-OHE, however, doubled during maximal exercise intensity. The 2-OHE:E and 2-MeOE:2-OHE ratios did not alter during incremental exercise. Training effects on acute exercise responses were only noticed for 4-OHE, which contrary to pre-training conditions, now progressively decreased. The major findings of this study are that in response to training: a) during rest, a greater proportion of CE are formed from a lower amount of precursor hormone, b) the rate of O-methylation of CE increases.

The present study was designed to assess the effects of acute exercise and short-term intensive training on catechol-O-methyltransferase (COMT) activity. COMT inactivates catecholamines and converts primary catecholestrogens (CE) into their O-methylated form yielding the 2- (2-MeOE) and 4-methoxyestrogens (4-MeOE). Blood samples were obtained from 15 previously untrained eumenorrheic women (mean +/- SE, VO2max: 43.8 mL x kg-1 x min-1 +/- 0.6) before and after a 5-d intensive training period, at rest and during incremental exercise. COMT activity was determined in the erythrocytes (RBC-COMT) after incubation of blood lysate with primary CE. The formation of both 2- and 4-MeOE was significantly higher (P < 0.05) during the luteal (LPh) than during the follicular phase (FPh). The amount of 2-MeOE formed (FPh: 4.2 +/- 0.2%; LPh: 4.9 +/- 0.2%) was significantly greater than the produced amount of 4-MeOE (FPh: 1.4 +/- 0.1%; LPh: 1.5 +/- 0.1%) (P < 0.05). Both before and after training, incremental exercise did not significantly alter RBC-COMT activity although we observed a trend for RBC-COMT activity increasing proportionally with the exercise intensity. After a brief period of exhaustive training, during rest the formation of 2-MeOE (FPh: +16.7%, LPh: +15.7%) and 4-MeOE (FPh: +28.6%; LPh: +40%) was significantly (P < 0.05) increased. The results of the present study are consistent with earlier findings reporting increased plasma concentrations of O-methylated CE following training. It is concluded that RBC-COMT activity is increased by brief intensive training, but not by acute exercise. We speculate that an increase in COMT-catalyzed O-methylation of CE may indicate that less COMT is available to deactivate norepinephrine.

Catecholestrogens (CE) represent a major metabolic pathway in estrogen metabolism. Previous information on CE and training is limited to two cross-sectional studies that did not involve standardized training. Our purpose, by means of a prospective design, was to evaluate the effects of a brief, exhaustive training program on resting plasma concentrations of 2-hydroxy CE. The experimental design spanned two menstrual cycles; a control cycle and a training cycle. The subjects were nine previously untrained, eumenorrheic women [body fat: 24.8 +/- 1.0 (SE) %]. Data were collected during the follicular (FPh) and the luteal phases (LPh). Posttraining FPh and LPh tests were held the day after the last day of a 5-day period of training on a cycle ergometer. Total 2-hydroxyestrogens (2-OHE) averaged 200 +/- 29 pg/ml during the FPh and 420 +/- 54 pg/ml during the LPh (P < 0.05). Levels of total 2-methoxyestrogens (2-MeOE) were 237 +/- 32 pg/ml during the FPh and 339 +/- 26 pg/ml during the LPh (P < 0.05). After training, although the plasma levels of 2-OHE significantly decreased (21%; P < 0.05) during the LPh, the actual CE formation (as estimated from the 2-OHE-to-total estrogens ratio) increased (+ 29%; P < 0.05). CE activity, as expressed by the 2-MeOE-to-2-OHE ratio, showed significantly higher values in both phases (FPh, + 14%; LPh, + 13%; P < 0.05). At the same time, resting levels of norepinephrine (NE) were increased by 42% (P < 0.05). CE strongly inhibit biological decomposition of NE by catechol-O-methyltransferase (COMT). Results of the present study suggest that, in response to training, CE are increasingly competing with the enzyme COMT, thus preventing premature NE deactivation.

It has been hypothesized that exercise-related hypo-estrogenemia occurs as a consequence of increased competition of catecholestrogens (CE) for catechol-O-methyltransferase (COMT). This may result in higher norepinephrine (NE) concentrations, which could interfere with normal gonadotropin pulsatility. The present study investigates the effects of training on CE responses to acute exercise stress. Nine untrained eumenorrheic women (mean percentage of body fat +/-SD: 24.8 +/- 3.1%) volunteered for an intensive 5-day training program. Resting, submaximal, and maximal (tmax) exercise plasma CE, estrogen, and catecholamine responses were determined pre- and post training in both the follicular (FPh) and luteal phase (LPh). Acute exercise stress increased total primary estrogens (E) but had little effect on total 2-hydroxyestrogens (2-OHE) and 2-hydroxyestrogen-monomethylethers (2-MeOE) (= O-methylated CE after competition for catechol-O-methyltransferase). This pattern was not significantly changed by training. However, posttraining LPh mean (+/-SE) plasma E, 2-OHE, and 2-MeOE concentrations were significantly lower (P < 0.05) at each exercise intensity (for 2-OHE: 332 +/- 47 vs. 422 +/- 57 pg/mL at tmax; for 2-MeOE: 317 +/- 26 vs. 354 +/- 34 pg/mL at tmax). Training produced opposite effects on 2-OHE:E ratios (an estimation of CE formation) during acute exercise in the FPh (reduction) and LPh (increase). The 2-MeOE:2-OHE ratio (an estimation of CE activity) showed significantly higher values at tmax in both menstrual phases after training (FPh: +11%; LPh: +23%; P < 0.05). After training, NE values were significantly higher (P < 0.05). The major findings of this study were that: training lowers absolute concentrations of plasma estrogens and CE; the acute exercise challenge altered plasma estrogens but had little effect on CE; estimation of the formation and activity of CE suggests that formation and O-methylation of CE proportionately increases. These findings may be of importance for NE-mediated effects on gonadotropin release.

Objective: To investigate the behavior of C4-substituted estrogens, the so-called catecholestrogens, in response to acute exercise and training. The 4-hydroxyestrogens are known to have both a strong estrogenic potency and affinity for catechol-O-methyltransferase (COMT), the enzyme that deactivates catecholamines., Design: A prospective trial covering three menstrual cycles: a control cycle, a moderate training cycle, and a heavy training cycle., Participant(s): Six untrained, healthy, eumenorrheic women (mean pretraining maximum oxygen uptake: 40.9 +/- 4.9 mL/kg per minute, body fat: 27.9% +/- 3.6%) volunteered for this study., Intervention(s): An incremental exercise test to exhaustion on a cycle ergometer, in the follicular and luteal phases, before and after a brief but exhaustive training program., Main Outcome Measure(s): Hormone measurements included follicular and luteal phase plasma E2, LH, catecholamines, PRL, total unconjugated and conjugated estrogens, total 4-hydroxyestrogens (4-OHE), and 4-hydroxyestrogen-monomethylethers (4-MeOE)., Result(s): Pretraining baseline 4-OHE levels were significantly higher in the luteal phase (66 +/- 9 pg/mL; mean +/- SEM) than in the follicular phase (51 +/- 7 pg/mL). Pretraining and post-training baseline 4-MeOE values were below minimal detection limits (< 35 pg/mL). During incremental exercise, catecholamines, PRL, E2, unconjugated and conjugated estrogens, 4-OHE, and 4-MeOE always increased (the increases in 4-OHE during exercise were more pronounced before training, contrary to the 4-MeOE being most increased after training). The baseline 4-MeOE:4-OHE ratio (a measure of catecholestrogen activity) significantly increased with progressive training., Conclusion(s): Because 4-OHE have been shown to be able to control the hypothalamic gonadotropin oscillator and to stimulate the luteolytic prostaglandin PGF2 alpha, the acute exercise-induced increases of 4-OHE and their positive correlation with lactate levels may indicate a key process in the pathogenesis of exercise-associated menstrual irregularities. In addition, 4-OHE, when insufficiently O-methylated, are known to be capable of raising mutagenic superoxide free radicals and causing DNA damage that may lead to breast cancer. The results of the present study also may be of significance for the apparent protective effects of sports participation against cancer of the breast.

Enzymatic O-methylation of catechol estrogens in red blood cells has been investigated with respect to species difference. In the presence of S-adenosylmethionine, 2- or 4-hydroxyestradiol (2-OHE2 or 4-OHE2) was incubated with blood lysate obtained from rats (five strains), guinea pigs, mice, rabbits, dogs, monkeys, and humans, respectively. The yielded guaiacols and unchanged substrate were determined by gas chromatography/mass spectrometry in a selected ion monitoring mode employing the corresponding 2H4-labeled compounds as internal standards. The total amounts of guaiacols formed from 2-OHE2 and 4-OHE2 were different, being the highest (79.6% and 38.1%) in monkeys and the lowest (5.1% and 1.9%) in humans. The ratios of isomeric guaiacols formed from 4-OHE2 (4Me/3Me) were 7.6-71, while those from 2-OHE2 (2Me/3Me) were 1.4-3.2. Thus, marked differences in O-methylation of catechol estrogens were observed among animal species, but no significant strain difference was detected in rats.

A simple and sensitive method for the determination of guaiaicol oestrogens enzymatically formed from 2- or 4-hydroxyoestradiol, by means of high-performance liquid chromatography with coulometric detection, has been developed. Catechol and guaiacol oestrogens were efficiently separated on a reversed-phase column, using 0.5% ammonium phosphate buffer (pH 3.0)-acetonitrile (59:41, v/v) as the mobile phase, and detected coulometrically in a screening-oxidation mode at +0.10 V and +0.35 V, respectively. The method was applied to the assay of in vitro enzymic O-methylation of catechol oestrogens. After 2- or 4-hydroxyoestradiol had been incubated with rat red blood cells in the presence of S-adenosylmethionine, the resulting guaiacols and unchanged substrate were percolated through an Extrelut-3 cartridge. The dried eluate was redissolved and directly injected. This simple procedure was as sensitive as the previously reported method using gas chromatography-mass spectrometry in a selected ion monitoring mode.

Catecholestrogens, 2-methoxyestrogens and "classical" estrogens (estrone, estradiol, estriol) were measured simultaneously in serum and urine samples of 220 pregnant women from the 8th week of pregnancy until to delivery. From these data we established the central 0.80 centile intervals as time specified reference intervals for each substance analyzed. Serum and urinary estradiol rise steadily during the progress of pregnancy, whereas estrone, catecholestrogens and 2-methoxyestrogens reach a plateau during the last trimester. These observations support the hypothesis, that the amount of the latter compounds may be regulated by separate mechanisms. The values of concentration and excretion of 2- and 4-substituted estrogens varied widely throughout pregnancy. Even very high or very low concentrations of these substances had no recognizable relation to the outcome of pregnancy. This supports the assumption that catecholestrogens and their methylethers are metabolites without any regulatory function in pregnancy.

To investigate the role of catechol estrogens in human parturition, these steroids were analyzed in samples from the maternal venous and umbilical venous and arterial plasma at vaginal (n = 28) and abdominal (n = 28) delivery. To ensure the appropriateness of collection of umbilical artery and venous blood samples, progesterone content was also determined. Although there is no significant difference in maternal vein content of catechol estrogens between the two groups, the umbilical venous (p = 0.03) and arterial (p = 0.002) plasma concentrations are significantly higher at vaginal delivery than those measured at abdominal delivery. In view of the present data and the importance of catechol estrogens in prostaglandin synthesis and in potentiating the activity of catecholamines through competitive inhibition of catechol-O-methyltransferase, it is suggested that catechol estrogens may play a role in triggering the events involved in the onset of labor and delivery in humans.

Competitive swimmers were followed over a 2-year period when they trained at different levels of exercise which coincided with distinct changes in their menstrual history. Oligomenorrhea was identified in 5 of 13 of these athletes when they swam approximately 100,000 yards per week. Weight and percentage of body fat were not significantly different between the period of oligomenorrhea and regular menstrual function (P = 0.24). Mean and median levels of luteinizing hormone, follicle-stimulating hormone, prolactin, and 17beta-estradiol were decreased and catechol estrogens and beta-endorphins were increased in serum during the strenuous, when compared with the moderate, training period. The serum levels of the steroid and protein hormones were similar to those of normal cycling, nonexercising control subjects during moderate exercise (60,000 yards per week). The significant differences between beta-endorphins and catechol estrogens during periods of strenuous exercise suggest an explanation for oligomenorrhea in female athletes. These hormonal changes result in hypothalamic anovulation, which appears to be reversible, because the hormone levels and menstrual cycles return to normal when the exercise is reduced.

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.

To provide a definite basis for studies on the biological effects of exogenously administered catecholestrogens, the time courses of the concentrations of these estrogens in serum, pituitary and CNS-tissues were studied in male rats after s.c. injection of either 150 microgram of 4-hydroxyestradiol or 2-hydroxyestradiol (dissolved in 200 microliter sesame oil/ethanol/ascorbic acid; 97.5/2.5/0.1; vol/vol/wt) or equimolar amounts of 4-hydroxyestradiol 3, 4-dibenzoate or 2-hydroxyestradiol 2,3-dibenzoate (dissolved in 200 microliter sesame oil). The injection of free catecholestrogens resulted in bolus-like elevations of the serum and tissue concentrations of the respective compound (max. values up to 9 ng/ml, half-life below 1 h) whereas the injection of catecholestrogen benzoates gave lower (max. values about 1 ng/ml) but prolonged elevations (half-life approx. 24 h and 32 h for 4-OHE2 and 2-OHE2) of the respective free catecholestrogen.

So as to evaluate metabolism of catecholestrogens during pregnancy, changes in plasma unconjugated estradiol (E2), estrone (E1), 2-methoxyestradiol (2-mE2) and 2-methoxyestrone (2-mE1) concentrations were measured after a bolus injection of 40mg of estrone-sulfate (E1-S) or 100mg of dehydroepiandrosterone-sulfate (DHA-S) to normal pregnant women near term. The injection of E1-S resulted in a marked rise in plasma 2-mE1 levels followed by a rapid marked increase in the plasma E1 concentration. The injection of DHA-S produced a small increase in the plasma 2-mE2 levels in spite of a marked increase in the plasma E2 concentration. No significant correlation between E2 and 2-mE2 was found, however, significant correlations between E1 and 2-mE1, between E1 and 2-mE2 and between 2-mE2 in in both groups were demonstrated. The marked increase in 2-mE1 compared to a small increase in 2-mE2 after DHA-S injection indicates that E2 may participate in the formation of catecholestrogen via its conversion to E1 during pregnancy. These results suggest that the main route for the formation of catecholestrogen is via E2----E1----(2OHE1)----2-mE1 in normal pregnancy near term.

A heterologous immunoassay for 2-hydroxyoestrogens has been established in which antibodies raised against 2-hydroxyoestradiol-17-succinyl-BSA serve as binding protein and 2-hydroxyoestrone-17-cmo-[125I]iodohistamine as radioligand. Lipophilic serum components competing for binding sites in this system were defined as total 2-hydroxyoestrogens'. The underlying assumption of specificity was supported by the pattern of cross-reactivity evaluated with structural related steroids and o-diphenols and by the fact, that an additional chromatography of the serum extracts preceding the competing reaction had little if any effect. Sensitivity: 2.8 +/- 1 pg/tube; accuracy: Y = 0.91x + 2.2; r = 0.989; precision: 5.8% intra-assay; 6.5% inter-assay. The following concentrations (+/- standard deviation) were found in the sera of healthy subjects. Young men: 29 +/- 5 pg/ml (n = 11); women follicular phase: 32 +/- 8 pg/ml (n = 25); luteal phase: 53 +/- 13 pg/ml (n = 23); postmenopausal women: 13 +/- 4 pg/ml (n = 10); pregnant women 11th--20th week: 70 +/- 16 mg/ml (n = 64); 36th--40th week: 240 +/- 23 pg/ml (n = 40); newborn cord blood: 604 +/- 43 pg/ml (n = 48).

Recently it has been attached importance to the physiological function of catecholestrogens. To elucidate it a specific radioimmunoassay of conjugated 2-hydroxyestrone (2-OHE1) and 2-hydroxyestradiol-17 beta (2-OHE2) in human plasma was attempted. After extracting with ethyl acetate, samples were purified by a short Sephadex LH-20 columnchromatography and determined using the antiserum to 2-OHE1-17(O-carboxymethyl) oxime-bovine serum albumin conjugate. The following results were obtained: 1) A long Sephadex LH-20 columnchromatography which was used for the purification of catecholestrogens produced chemically, had a superior faculty in separation and high capacity 2) The antiserum cross-reacted 26.4% with 1-OHE2, but less than 1% with other steroid hormones. 3) The sensitivity of the method was around 10pg in both 2-OHE1 and 2-OHE2 assays. The method blank determined using 2ml plasma of bilaterally adreno-oophrectomized women was below 15 pg/ml in both 2-OHE1 and 2-OHE2 assays (n = 24). 4) The coefficient of variation in both accuracy and between-assay precision of the method was less than 17%. 5) The plasma 2-OHE1 concentration was below 15 pg/ml in normal men (n = 8) and non-pregnant women (n = 13). The concentration in pregnant women was 20 +/- 8 pg/ml (SD, n = 7) in 1st trimester of pregnancy, 58 +/- 13 (n = 3) in 2nd trimester and 177 +/- 66 (n = 12) in 3rd trimester. The E1/2-OHE1 and E2/2-OHE1 ratios in 3rd trimester of 9 pregnant women were 19.7 +/- 11.9(SD) and 78.0 +/- 27.8, respectively. 6) The plasma 2-OHE2 concentration was below 15 pg/ml in normal men (n = 8), non-pregnant women (n = 8), and 1st to 2nd trimester (n = 10) and 15 +/- 9 pg/ml (SD, n = 13) in 3rd trimester of pregnant women.