Potential role of ultra-sensitive estradiol assays in estimating the risk of breast cancer and fractures

Radioimmunoassays (RIA) for the measurement of estradiol are sufficiently sensitive to assess the reproductive status of pre-menopausal women but lack sufficient sensitivity for low concentrations found in post-menopausal women. Bioassays have been used in the past to measure low estrogen levels but are impractical for handling high volumes of tests, particularly routine and non-research specimens. In this study, we compared results for estradiol using several different methods including bioassay, RIA, and two tandem mass spectrometry methods. At the lower tertile of estradiol measurements by RIA, the overall excellent correlation with results obtained by tandem mass spectrometry (i.e. r=0.83) was lost (i.e. r=0.29). In addition, results were much lower with bioassay and mass spectrometry than with RIA suggesting that RIA measures undesired noise or estrogen metabolites. The mass spectrometry methods correlate best with isotopic kinetic methods when assessing aromatase inhibition. On this basis, we conclude that mass spectrometry assays are the best option for measurement of low estradiol concentrations. With such assays, greater discrimination should be achievable when using estradiol levels as a predictor of the risks for breast cancer and for fractures.     

Role of steroid sulfatase in local formation of estrogen in post-menopausal breast cancer patients

More than two-thirds of breast cancers occur in post-menopausal women, and depend on the estrogens for their proliferation and survival. For the treatment of estrogen-dependent breast cancers, two major treatment options are now available. One is selective estrogen receptor modulator (SERM) such as Tamoxifen and another is aromatase inhibitor such as Anastrozole, Letrozole and Exemestane, which reduce local in situ formation of estrogens. Although these therapies are clinically active for advanced and early breast cancers, de novo and/or acquired resistance to SERM and/or aromatase inhibitors are also clinical problem. Recent studies suggest that local formation of estrogens in the breast tumors is more important than circulating estrogen in plasma for the growth and survival of estrogen-dependent breast cancer in post-menopausal women. The rationale for the importance of local formation of estrogens is based on the following evidences. Estradiol (E2) levels in breast tumors are equivalent to those of pre-menopausal patients, although plasma E2 levels are 50-fold lower after menopause. E2 concentrations in breast tumors of post-menopausal women are 10–40 times higher than serum level. Biosynthesis of estrogens in breast tumors tissues occurs via two major different routes, one is aromatase pathway and another is steroid-sulfatase (STS) pathway. Whereas many studies has been reported about aromatase inhibitor and its clinical trial results in breast cancer patients, limited information are available regarding to other estrogen regulating enzymes including STS, its role in breast tumors and STS inhibitors. STS is the enzyme that hydrolyses estrone 3-sulfate (E₁S) and dehydroepiandrosterone-sulfate (DHEA-S) to their active un-sulfoconjugated forms, thereby stimulating the growth and survival of estrogen-dependent breast tumors. It has been well known that E₁S level are much higher than E2 level both in plasma and tumor of post-menopausal patients. Recent reports show that more than 80% of breast tumors are stained with anti-STS antibody and the expression of STS is an independent prognostic factor in breast cancer. Taking these findings into consideration, local formation of estrogens could be partially synthesized from large amount of E₁S by STS, which exist in breast cancer. On the other hand, aromatase localizes in stroma and adipocyte surrounding breast cancer. Furthermore, since estrogen formation from E₁S and DHEA-S (STS pathway) cannot be blocked by aromatase inhibitors, STS is thought to be a new molecular target for the treatment of estrogen-dependent tumor post-SERM and/or aromatase inhibitors. In this symposium, these recent rationale for the importance of STS in post-menopausal breast cancer patients is reviewed as well as STS inhibitor.     

The Sulawesi Crested Black Macaque (Macaca nigra) menstrual cycle: changes in perineal tumescence and serum estradiol, progesterone, follicle-stimulating hormone, and luteinizing hormone levels.

Events in the normal menstrual cycle of the endangered Sulawesi Crested Black Macaque (Macaca nigra) were characterized. Daily blood samples were obtained during 10 menstrual cycles from five M. nigra demonstrating regular cycles. The amount of perineal tumescence was scored daily. Serum levels of estradiol and progesterone were determined by RIA, serum LH levels were determined by the mouse Leydig cell bioassay, and serum FSH levels were determined by the rat granulosa cell aromatase bioassay. Cycle length was 39.8 +/- 1.0 days (mean +/- SEM) with an LH surge occurring 25 +/- 1.5 days from the onset of menses. After menses, both LH and estradiol were initially depressed, with estradiol first exceeding 50 pg/ml 8 days before the LH surge. In five cycles, peak estradiol levels (340 +/- 44 pg/ml) occurred on the day of the LH surge (637 +/- 58 ng/ml) and in the other five cycles, on the day before the LH surge. There was a broad increase of FSH in midcycle without a well-defined surge corresponding to the LH surge. Progesterone began increasing on the day of the LH surge and reached peak levels (6.8 +/- 0.96 ng/ml) 8 days later. Maximal perineal tumescence was generally associated with the time of the LH surge, but variation between animals made it impossible to predict accurately the day of the LH surge by perineal tumescence scores alone.     

The role of estrogen in bone growth and maturation during childhood and adolescence

Twenty years ago it was believed that pubertal growth was stimulated by testicular androgen in boys and by adrenal androgen in girls. Estrogen, which was used to inhibit growth in excessively tall girls, was not thought to have growth-promoting effects. We hypothesized that estrogen has a biphasic effect on epiphyseal growth, with maximal stimulation at low levels. We showed that the administration of low doses of estrogen, corresponding to a serum estradiol level of about 4 pg/ml (15 pmol/l) caused more than a 60% increase over the prepubertal growth rate in both boys and girls. To test the hypothesis that estrogen is the principal mediator of the pubertal growth spurt in boys, we administered the aromatase inhibitor, testolactone, to boys with familial male-limited precocious puberty. Testolactone produced near normalization of both growth velocity and bone maturation, despite levels of serum testosterone that remained within the adult male range. The observation that low levels of estrogen stimulate growth and bone maturation suggested that estrogen might explain the more rapid epiphyseal maturation of prepubertal girls compared to boys. To determine whether prepubertal girls have higher estrogen levels than prepubertal boys, we developed an ultrasensitive recombinant cell bioassay for estrogen with a sensitivity of 0.02 pg/ml (0.07 pmol/l) estradiol equivalents. Prepubertal girls had approximately eight-fold higher levels of serum estradiol than did prepubertal boys (0.6 ± 0.6 pg/ml (SD) (2.2 ± 2.2 pmol/l) vs 0.08 ± 0.2 pg/ml (0.29 ± 0.73 pmol/l), P < 0.05). We concluded that the pubertal growth spurt of both sexes is driven primarily by estrogen, and that the more rapid epiphyseal maturation of prepubertal girls (vs boys) may be explained by their higher estradiol levels.     

Biological effects of hormone replacement therapy in relation to serum estradiol levels

OBJECTIVE: Tissues in various parts of the body have different sensitivities to estradiol. However, it is very difficult to measure the serum estradiol levels precisely in women receiving oral conjugated equine estrogen, which is a mixture of estrogens. In the present study, we precisely measured the serum levels of estradiol in postmenopausal women undergoing hormone replacement therapy (HRT), and we clarified the relationships between serum estradiol levels and the effects of HRT on the Kupperman index, bone mineral density (BMD), serum gonadotropin, lipid metabolism and unscheduled bleeding as the clinical endpoints. METHODS: Sixty-eight postmenopausal or bilaterally ovariectomized women, aged 30-64 years, who had been suffering from vasomotor symptoms such as hot flush or atrophy of the vagina were randomly assigned to two groups: one group of 34 patients who received oral administration of 0.625 mg conjugated equine estrogen (CEE, Premarin, Wyeth) and 2.5 mg medroxyprogesterone acetate (MPA, Provera, Upjohn) every other day, and another group of 34 patients who received oral administration of 0.625 mg CEE and 2.5 mg MPA every day. All subjects were re-classified into three groups according to the serum estradiol level after 12 months of treatment: (1) low estradiol group (<15 pg/ml, n = 25); (2) middle estradiol group (> or =15 and <25 pg/ml, n = 27), and (3) high estradiol group (> or =25 pg/ml, n = 16). We examined the relationships between serum estradiol level and the effects of estradiol on the Kupperman index, BMD, serum gonadotropin levels, lipid profile and unscheduled bleeding in these three groups. Results: Results obtained by using our newly developed high-performance liquid chromatography (HPLC)-radioimmunoassay (RIA) system clearly showed that the effects on each tissue in postmenopausal women receiving oral CEE and MPA is closely related to estradiol level. The effects of HRT on BMD, serum gonadotropin levels and lipid profile were shown to be clearly dependent on the serum estradiol levels, while the effect of HRT on the Kupperman index was independent of the serum estradiol level. Furthermore, it was also found that a very low concentration of estradiol (<15 pg/ml) was sufficient to suppress the serum LH and FSH levels and to relieve vasomotor symptoms, and that the minimum concentration of estradiol required to increase BMD was 15 pg/ml. On the other hand, the level of estradiol required to reduce total cholesterol, low density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (Apo B) was found to be more than 25 pg/ml, while the level required to increase high density lipoprotein-cholesterol (HDL-C) and apolipoprotein A1 (Apo A1) was at least 15 pg/ml. The incidence of unscheduled bleeding was also lower in the low estradiol group than in the other estradiol level groups. CONCLUSION: These results suggest that the different clinical endpoints have different response thresholds and thus reflect tissue sensitivity to estradiol levels achieved by HRT.     

The potential role of estrogen in aromatase regulation in the breast

Aromatase is expressed in both normal and malignant breast tissues. Aromatase activity in the breast varies over a wide range. Our previous studies have demonstrated that in situ aromatization contributes to the estrogen content of breast tumors to a major extent. Consequently, alterations of aromatase activity could serve as a major determinant of tissue estradiol content. However, the mechanisms and extent of aromatase regulation in breast tissues have not been fully established. We have observed an inverse correlation between tumor aromatase activity and estrogen content in nude mice bearing xenografts of MCF-7 cells transfected with the aromatase gene. To investigate the potential role of estrogen in aromatase regulation in the breast, studies were carried out in an in vitro model. In this model, MCF-7 cells were cultured long term in estrogen-deprived medium and called by the acronym, LTED cells. We found that long-term estrogen deprivation enhanced aromatase activity by 3–4-fold when compared to the wild-type MCF-7 cells. Re-exposure of LTED cells to estrogen reduced aromatase activity to the levels of the wild-type MCF-7 cells. We also measured aromatase activity in 101 frozen breast carcinoma specimens and compared tumor aromatase activities in pre-menopausal patients versus post-menopausal patients and in post-menopausal patients with or without hormone replacement therapy (HRT). Although statistically not significant, there was a trend paralleling that observed in the in vitro studies. Aromatase activity was higher in breast cancer tissues from the patients with lower circulating estrogen levels. Our data suggest that estrogen may be involved in the regulation of aromatase activity in breast tissues.     

Lipid peroxidation at various estradiol concentrations in human circulation during ovarian stimulation with exogenous gonadotropins.

The aim of our study was to investigate the correlation between serum malondialdehyde levels and serum estradiol concentrations in healthy human female subjects. Nine hundred and fifty-five blood samples, from infertile women undergoing controlled ovarian hyperstimulation treatment with recombinant follicle-stimulating hormone, were collected for estradiol and malondialdehyde measurements. Five groups were formed according to serum estradiol levels: Group I (< 50 pg/ml), group II (50 - 299 pg/ml), group III (300-999 pg/ml), group IV (1000-1999 pg/ml) and group V (> or = 2000 pg/ml). One-way analysis of variance was used for comparisons. Mean malondialdehyde concentrations were 1.74 +/- 0.24 mmol/ml (group I), 1.53 +/- 0.20 mmol/ml (group II), 1.69 +/- 0.24 mmol/ml (group III), 1.77 +/- 0.21 mmol/ml (group IV) and 1.86 +/- 0.20 mmol/ml (group V), respectively. Mean serum malondialdehyde level at physiological estradiol concentrations (50-199 pg/ml, group II) was significantly (p < 0.01) lower than the mean malondialdehyde levels in other groups. Mean malondialdehyde concentrations among the remaining groups did not significantly differ. Our findings suggest that in vivo lipid peroxidation might be increased when circulating estradiol concentrations are below (< 50 pg/ml) or above (> 300 pg/ml) the physiological limits. High blood estradiol levels in human female subjects during ovarian stimulation with exogenous gonadotropins could be associated with increased serum malondialdehyde concentrations.     

Plasma estrogen suppression with aromatase inhibitors evaluated by a novel, sensitive assay for estrone sulphate

Aromatase inhibition is a well-defined treatment option for postmenopausal breast cancer. Although several aromatase inhibitors such as aminoglutethimide, formestane, fadrozole have been found to inhibit in vivo aromatization by>85%, previous studies reported plasma estrogen levels to be sustained at approximately 20–50% of their control level during treatment with these drugs. The discrepancy could be due to lack of sensitivity or non-specific crossreactions in the radioimmunoassay (RIA) methods. Mean plasma levels of estrone (E₁) and estradiol (E₂) in postmenopausal women are approximately 80 and 20 pmol/l, respectively; on the contrary, mean plasma levels of the estrogen conjugate estrone sulphate (E₁S) are approximately 4–500 pmol/l. Most RIA methods for plasma E₂ and E₁ measurements have sensitivity limits in the range of 2–3 and 7–10 pmol/l, respectively; accordingly, the suppression of plasma estrogens by more than 80–90% will produce hormone values below the sensitivity limit of the method in many patients. Recently, we developed a new method to determine plasma E₁S. This assay has a sensitivity limit of 2.7 pmol/l. In theory, this method may allow the determination of plasma E₁S levels suppressed to less than 2% of control values in the majority of patients. Using this method, we found different aromatase inhibitors such as formestane, aminoglutethimide, formestane and aminoglutethimide administered in concert or anastrozole to suppress plasma E₁S levels down to 24, 13, 7 and 4%, respectively. The suppression of plasma E₁S evaluated with this method thus approaches the percentage aromatase inhibition measured with tracer studies.     

Does alligator testis produce estradiol? A comparison of ovarian and testicular aromatase

Testicular secretion of estradiol is necessary for normal spermatogenesis and male reproductive physiology in humans and rodents. The role of estradiol in nonmammalian vertebrates remains unknown, but elevated circulating estradiol has been reported in male lizards, alligators, and various bird species. We have been unable to detect circulating estradiol in male alligators; therefore, we reexamined the question of testicular production of estradiol in alligators using more rigorous assay procedures. A large pool of plasma from a male alligator was extracted and run through an HPLC column. Immunoreactive estradiol-like material eluted coincident with authentic estradiol. By using an ultrasensitive RIA and processing large volumes of male plasma (1000 microl), we were able to measure estradiol. Estradiol in male alligators ranged from 0.23 to 3.14 pg/ml, whereas estradiol in immature female alligators ranged from 14 to 66 pg/ml. Aromatase activity in microsomes from adult alligator ovarian tissue was 36.2 +/- 1.6 pmol mg-1 h-1, whereas activity in testicular microsomes ranged between 0.92 and 2.38 pmol mg-1 h-1. Ovarian aromatase activity was inhibited in a concentration-dependent fashion by Fadrozole, but the essentially background activity of testicular aromatase was not inhibited at any concentration of Fadrozole. Likewise, a comparison of alligator testicular and ovarian aromatase mRNA expression gave a similar result: the ovarian expression was 600-fold higher and brain tissue was 10-fold higher than that of the testis. Circulating estradiol in male alligators is probably of extragonadal origin, and the testis produces little if any of this steroid.     

Gender, exercise training, and eNOS expression in porcine skeletal muscle arteries.

Our purpose was to determine the effects of gender and exercise training on endothelial nitric oxide synthase (eNOS) and superoxide dismutase (SOD) protein content of porcine skeletal muscle arteries and to evaluate the role of 17beta-estradiol (E2) in these effects. We measured eNOS and SOD content with immunoblots and immunohistochemistry in femoral and brachial arteries of trained and sedentary male and female pigs and measured estrogen receptor (ER) mRNA and alpha-ER and beta-ER protein in aortas of male and female pigs. Results indicate that female arteries contain more eNOS than male arteries and that exercise training increases eNOS content independent of gender. Male and female pigs expressed similar levels of alpha-ER mRNA and protein and similar amounts beta-ER protein in their arteries. E2 concentrations as measured by RIA were 180 +/- 34 pg/ml in male sera and approximately 5 pg/ml in female sera, and neither was changed by training. However, bioassay indicated that biologically active estrogen equivalent to only 35 +/- 5 pg/ml was present in male sera. E2 in female pigs, whether measured by RIA or bioassay, was approximately 24 pg/ml at peak estrous and 2 pg/ml on day 5 diestrus. The free fraction of E2 in sera did not explain the low measurements, relative to RIA, of E2. We conclude that 1). gender has significant influence on eNOS and SOD content of porcine skeletal muscle arteries; 2). the effects of gender and exercise training vary among arteries of different anatomic origin; 3). male sera contains compounds that cause RIA to overestimate circulating estrogenic activity; and 4). relative to human men, the male pig is not biologically estrogenized by high levels of E2 reported by RIA, whereas in female pigs E2 levels are lower than in the blood of human women.     

Hormonal effects of aromatase inhibitors: focus on premenopausal effects and interaction with tamoxifen

Third generation aromatase inhibitors have excellent specificity. Some reports indicate that letrozole may have a minor effect on cortisol synthesis but these were not confirmed: valid comparisons with other aromatase inhibitors requires randomised study.

The putative use of a third generation inhibitor as a single agent in premenopausal women has been investigated using YM511. It was hypothesised that in this situation site-specific suppression of estrogens in breast carcinomas, without systemic effects, may lead to a down-regulation of tumour proliferation. Plasma levels of androstenedione and testosterone were significantly increased by 2 weeks treatment with YM511. Mean plasma estrone levels were suppressed, but some plasma estradiol levels were abnormally high and others abnormally low. These differential effects of YM511 on circulating estrogens supported the concept that peripheral synthesis of estrogens might be suppressed while ovarian production remained high. However, YM511 did not demonstrate anti-proliferative effects in hormone sensitive breast carcinomas.

Consideration of the pharmacology of the estrogen receptor during tamoxifen therapy indicates that tamoxifen effectively saturates the receptor (>99.94% occupancy) in postmenopausal women. The addition of an aromatase inhibitor in this situation would be very unlikely to affect the biological activity of the estrogen receptor. This provides a possible explanation why the clinical efficacy of tamoxifen combined with an aromatase inhibitor appears to be equivalent to that of tamoxifen alone.     

Estrone sulfate and dehydroepiandrosterone sulfate concentrations in normal subjects and men with cirrhosis.

Circulation levels of estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHAS) have been measured in plasma using a radioimmunoassay for estrone and dehydroepiandrosterone following extraction and hydrolysis of the sulfate. The mean +/- SE concentrations of E1S and DHAS in normal men were 458 +/- 25 pg/ml and 1.45 +/- 0.19 micrograms/ml, respectively. In normal women the values for days 5-7 of the cycle were 880 +/- 117 pg/ml and 1.25 +/- 0.12 micrograms/ml which were not different than the values for days 20-22 of 1195 +/- 176 pg/ml and 1.58 +/- 0.29 micrograms/ml. The mean values in post-menopausal women were 250 +/- 33 pg/ml and 0.47 +/- 0.07 micrograms/ml, both lower than the values in young women. In a group of cirrhotic men the mean values were 325 +/- 55 pg/ml and 0.38 +/- 0.12 micrograms/ml, both significantly lower than the normal values. This suggests a defect in sulfurylation in men with hepatic cirrhosis.     

Aromatase inhibitors: basic and clinical studies

Application of aromatase inhibitors to the treatment of conditions in which estrogen plays, a role is discussed. Studies in vitro demonstrate that 4-hydroxyandrostenedione (4-OHA) is a potent inhibitor of aromatase. The compound reduces ovariant estrogen production and causes regression of carcinogen (DMBA)-induced mammary tumors in the rat. In the rhesus monkey, 4-OHA was also shown to inhibit peripheral aromatization. To date 58 postmenopausal breast cancer patients with advanced metastatic disease have received 500 mg im weekly while 31 patients received 250 mg 4-OHA orally per day. Estradiol levels were significantly reduced in all patients from a mean of 7.2 + 0.8 pg/ml to 2.8 + 0.3 pg/ml. Of patients receiving 4-OHA im 27% had partial or complete responses and in 10% of patients the disease was stabilized. Similar responses occurred in the patients receiving 4-OHA orally. These results suggest that 4-OHA is effective and that this compound and other aromatase inhibitors could be valuable new additions to the treatment of breast cancer.     

Subnormal follicular-phase serum progesterone levels and elevated follicular-phase serum estradiol levels in young women with insulin-dependent diabetes

In a search for possible hormonal reasons for the loss of protection from myocardial infarction seen in diabetic women, serum levels of estradiol, progesterone, and luteinizing hormone were compared throughout a menstrual cycle (17 points) in eight healthy nonsmoking women and five otherwise healthy nonsmoking insulin-dependent diabetic women. The total length of the menstrual cycle and the lengths of the follicular and luteal phases did not differ between the groups. During the periovulatory and luteal phases, there was no significant intergroup difference with respect to any of the three hormones. During the follicular phase, in both groups, there was a plateau in serum progesterone concentration, with the level approximately 42% lower in the diabetic group (12.0 +/- 6.6 ng/dl versus 20.7 +/- 5.7; P less than 0.0001). Follicular-phase serum estradiol showed a rising curve in both groups; day-by-day comparison (days -10 to -3 before the luteinizing hormone peak) showed consistently higher levels in the diabetic group (mean, 108 pg/ml versus 95 pg/ml; P less than 0.001). The follicular-phase serum estradiol to progesterone ratio was nearly twice as high in the diabetic group as in the normal group (8.9 versus 4.6), a difference that was highly significant. The finding of elevated serum estradiol and subnormal serum progesterone concentrations during the follicular phase is so far unique to women with insulin-dependent diabetes mellitus. The possibility that this pronounced abnormality in diabetic women may be related to coronary disease merits testing in suitable in vivo and in vitro models of atherogenesis.     

The effect of smoking on serum progesterone, estradiol, and luteinizing hormone levels over a menstrual cycle in normal women

Since smoking has been shown to affect serum progesterone and estradiol levels in postmenopausal women, we evaluated the levels of these hormones and luteinizing hormone (LH) over an entire menstrual cycle (17 points) in eight healthy nonsmokers and eight healthy smokers. The total length of the cycle and the lengths of the follicular and luteal phases did not differ between the groups. There was no difference in estradiol, progesterone, or LH levels during the periovulatory and luteal phases. Follicular-phase serum progesterone, which had a level 37% higher in smokers, showed a plateau in both groups (28.3 +/- 5.7 ng/dl versus 20.7 +/- 5.7; P less than 0.0001). Follicular-phase serum estradiol showed a rising curve in both groups. The mean value in smokers was slightly higher than that in nonsmokers (107 pg/ml versus 95; P approximately 0.05); during the early part of the follicular phase, prior to the rapid preovulatory increase, the difference was greater (23%) and of higher statistical significance (80 pg/ml versus 65; P less than 0.001). The follicular-phase LH levels of smokers were skewed downward from the levels in nonsmokers, presumably by negative feedback from the elevated estradiol and progesterone levels; the difference was significant (P less than 0.001). The elevations of serum progesterone and estradiol in smokers probably represent activation of adrenocortical secretion by smoking. The greater and more clear-cut rise of progesterone than of estradiol is probably due to the fact that essentially all of the follicular-phase serum progesterone is secreted by the adrenal, while only part of the follicular-phase serum estradiol comes from the adrenal (via androstenedione and estrone).     

Ghrelin is present in human colostrum, transitional and mature milk

Ghrelin and its mRNA have recently been found in numerous human tissues including breast. The aim of this study was to compare the ghrelin levels in colostrum, mature and transitional milk and plasma in lactating women with plasma samples from non-lactating women. Venous blood samples were obtained from 17 healthy lactating women aged 22-35 years and from 16 age-matched controls. Colostrum, transitional and mature milk samples were collected just before suckling. The level of bioactive ghrelin was determined by RIA. Comparison of ghrelin values for lactating women showed significantly lower concentrations in colostrum (70.3 +/- 18 pg/ml), transitional milk (83.8 +/- 18pg/ml) and mature milk (97.3 +/- 13 pg/ml) than in the corresponding plasma samples (first day 95 +/- 16 pg/ml, 10th day 111 +/- 13 pg/ml and 15th day 135 +/- 16 pg/ml). The plasma concentrations were lower in the lactating than in the non-lactating women. Thus, the ghrelin levels in colostrum, transitional and mature milk were elavated concomitantly with increasing plasma ghrelin after delivery. The origin of milk ghrelin is not known, but it probably comes from the plasma.     

Breast cancer tissue estrogens and their manipulation with aromatase inhibitors and inactivators

Despite the dramatic fall in plasma estrogen levels at menopause, only minor differences in breast tissue estrogen levels have been reported comparing pre- and postmenopausal women. Thus, postmenopausal breast tissue has the ability to maintain concentrations of estrone (E₁) and estradiol (E₂) that are 2–10- and 10–20-fold higher than the corresponding plasma estrogen levels. This finding may be explained by uptake of estrogens from the circulation and/or local estrogen production. Local aromatase activity in breast tissue seems to be of crucial importance for the local estrogen production in some patients while uptake from the circulation may be more important in other patients. Beside aromatase, breast tissue expresses estrogen sulfotransferase and sulfatase as well as dehydrogenase activity, allowing estrogen storage and release in the cells as well as conversions between estrone and estradiol. The activity of the enzyme network in breast cancer tissue is modified by a variety of factors like growth factors and cytokines. Aromatase inhibitors have been used for more than two decades in the treatment of postmenopausal metastatic breast cancer and are currently investigated in the adjuvant treatment and even prevention of breast cancer. Novel aromatase inhibitors and inactivators have been shown to suppress plasma estrogen levels effectively in postmenopausal breast cancer patients. However, knowledge about the influence of these drugs on estrogen levels in breast cancer tissue is limited. Using a novel HPLC-RIA method developed for the determination of breast tissue estrogen concentrations, we measured tissue E₁, E₂ and estrone sulfate (E₁S) levels in postmenopausal breast cancer patients before and during treatment with anastrozole. Our findings revealed high breast tumor tissue estrogen concentrations that were effectively decreased by anastrozole. While E₁S was the dominating estrogen fraction in the plasma, estradiol was the estrogen fraction with the highest concentration in tumor tissue. Moreover, plasma estrogen levels did not correlate with tissue estrogen concentrations. The overall experience with aromatase inhibitors and inactivators concerning their influences on breast tissue estrogen concentrations is summarized.     

Estrone sulfate, estrone and estradiol concentrations in normal and cirrhotic postmenopausal women

Circulating levels (mean +/- SD) of estrone sulfate (E1S), estrone (E1) and estradiol-17 beta (E2) were measured in normal and cirrhotic postmenopausal women matched for body weight and age. In cirrhotic postmenopausal women, the E1S concentrations (201 +/- 46 pg/ml), while both E1 and E2 levels showed an increase (46 +/- 7 and 30 +/- 8 pg/ml) compared to control subjects (32 +/- 6 and 18 +/- 7 pg/ml). These data suggest that the liver plays an important role on the control of estrogen sulfation.     

Changes in profiles of serum sex steroids of male buffaloes from birth to maturity

Age-related changes in testosterone, progesterone and estradiol 17-beta were determined by radioimmunoassay (RIA) in the serum of 155 male buffalo calves of varying ages. The calves were classified into 17 age groups. The mean weight of calves increased from 33.6 +/- 9.6 kg at one week of age to 531 +/- 41.4 kg at 42 months. The testosterone levels were less than 100 pg/ml from birth until 15 months of age, followed by peak concentrations of 422 +/- 79 pg/ml at 24 to 30 months and 793 +/- 193 pg/ml at 42 to 48 months (corresponding to puberty and maturity, respectively). The progesterone levels were higher in newly born calves and mature bulls. Otherwise, the levels continued to be low throughout the period of growth and development. Estradiol 17-beta was significantly higher in postnatal calves up to two months of age. The testosterone revealed a positive correlation with weight and age while E2 17-beta showed a negative correlation with age. These results do not support a direct role of peripheral progesterone and estradiol 17-beta in the onset of puberty and sexual maturity of buffalo bulls.