Postmenopausal Plasma Research Articles

Clinical association of progesterone receptor isoform A with breast cancer metastasis consistent with its unique mechanistic role in preclinical models

BackgroundLuminal breast cancer (L-BCa) comprises the majority of incurable, distally metastatic breast cancer cases. Estrogen supports growth of L-BCa cells but suppresses invasiveness. Estrogen also induces the progesterone receptor (PR). Invasiveness and metastasis of L-BCa cells is supported by the short PR isoform (PR-A), in response to the range of pre- and post-menopausal plasma hormone levels, by counteracting the effects of estrogen via micro RNA-mediated cross-talk with the estrogen receptor (ER). PR-B directly supports L-BCa invasion and metastasis and also inhibits tumor growth, both only at high progesterone levels. As public datasets on L-BCa tumors cannot distinguish PR-A, this study was designed to seek clinical evidence for the role of PR-A in metastasis in comparison with PR-B and ER.MethodsMeasurement of tumor PR-A, PR-B and ER mRNA expression in 125 treatment-naive primary L-BCa patients with differential node involvement and analysis using linear mixed effects models. Transcriptional activity assays of PR-A and PR-B.ResultsLymph node involvement was strongly associated with PR-A expression (median, 3-fold higher vs. node-negative), independent of age, pathologic type, tumor grade, HER2 and PR-B. PR-B and ER correlated weakly with PR-A, but whereas PR-B and the PR-A/PR-B ratio were not significantly associated with node involvement, ER weakly negatively correlated with node positivity. PR-A was hypersensitive to mifepristone compared with PR-B.ConclusionsTaken together with previous mechanistic studies, the findings provide clinical evidence in support of the role of PR-A in L-BCa metastasis. They also suggest the possibility of developing selective PR-A modulators for future interventions in appropriate clinical situations.

Liquid chromatography-tandem mass spectrometric analysis of ten estrogen metabolites at sub-picogram levels in breast cancer women.

The measurement of estrogens at sub-picogram levels is essential for research on breast cancer and postmenopausal plasma. Heretofore, these concentration levels have rarely been achieved. However, it is possible through derivatization but still represent problems for monitoring catechol estrogens and 16α-hydroxyestrone (16α-OH-E1). Estrogens possess poor ionization efficiency in MS/MS, which results in insufficient sensitivity for analyzing samples at trace concentrations. The method presented here was used to extract ten estrogen metabolites (EMs) with a derivatization step involving a new adduct. The electrospray ionization (ESI) MS/MS sensitivity for the EMs was enhanced by derivatization with 3-bromomethyl-propyphenazone (BMP). The lower limits of quantification (LLOQ) of the EMs were 12-100 femtogram on-column, equivalent to 0.3-3.6pg/mL plasma, and the limits of detection (LOD) were 0.1-0.8pg/mL plasma. The percentage coefficient of variation (CV%) at the LLOQ was <20 for all investigated EMs. Ionization suppression was minimized by reacting the excess reagent, BMP, with methanol. The method was successfully applied for the determination of ten EMs in the plasma of fifty healthy postmenopausal and fifty ductal breast cancer women aged 47-65 years old. 16α-OH-E1 and three catechol estrogen metabolites, 4-OH-E1, 2-OH-E2 and 4-OH-E2, were successfully measured in the plasma of healthy and breast cancer women. The methyl-propyphenazone-EM derivatives exhibited better sensitivity in ESI-MS (7.5-fold) compared to the commonly used dansylation procedure.

Role of the short isoform of the progesterone receptor in breast cancer cell invasiveness at estrogen and progesterone levels in the pre- and post-menopausal ranges.

Overexpression of the progesterone receptor (PR) isoform A (PR-A) is a negative prognosticator for estrogen receptor (ER)-positive breast cancer but in vitro studies have implicated PR-B in progestin-induced invasiveness. As estrogen is known to suppress invasiveness and tumor progression and as the in vitro studies were conducted in models that either lacked ER or excluded estrogen, we examined the role of PR isoforms in the context of estrogen signaling. Estrogen (< 0.01nM) strongly suppressed invasiveness in various ER+ model cell lines. At low (< 1nM) concentrations, progestins completely abrogated inhibition of invasiveness by estrogen. It was only in a higher (5 nM - 50 nM) concentration range that progestins induced invasiveness in the absence of estrogen. The ability of low dose progestins to rescue invasiveness from estrogen regulation was exclusively mediated by PR-A, whereas PR-B mediated the estrogen-independent component of progestin-induced invasiveness. Overexpression of PR-A lowered the progestin concentration needed to completely rescue invasiveness. Among estrogen-regulated genes, progestin/PR-A counter-regulated a distinctive subset, including breast tumor progression genes (e.g., HES1, PRKCH, ELF5, TM4SF1), leading to invasiveness. In this manner, at relatively low hormone concentrations (corresponding to follicular stage and post-menopausal breast tissue or plasma levels), progesterone influences breast cancer cell invasiveness by rescuing it from estrogen regulation via PR-A, whereas at higher concentrations the hormone also induces invasiveness independent of estrogen signaling, through PR-B. The findings point to a direct functional link between PR-A and progression of luminal breast cancer in the context of the entire range of pre- and post-menopausal plasma and breast tissue hormone levels.

Abstract 5038: Role of the short isoform of the progesterone receptor in hormone-regulated invasiveness of breast cancer cells at pre- and post-menopausal levels of estrogen and progesterone

Abstract Introduction: High expression of the progesterone receptor (PR) isoform A, relative to PR isoform B, is clinically associated with progression to invasiveness in estrogen receptor (ER)-positive breast cancer and lower disease free survival. This is apparently at odds with in vitro studies implicating PR-B in mediating progestin-induced invasiveness. As the in vitro studies were conducted in the absence of estrogen (E2) signaling, we considered the possibility that modulation of E2 action may comprise a distinct aspect of the regulation of invasiveness by progestins. Methods: Standard ER+/PR+ and ER+/PR+/HER2+ breast cancer cell line models were studied, as well as recombinant cells expressing a single PR isoform. For the progestin hyper-sensitization studies, PR-A was over-expressed by lentiviral infection. Hormonal influence on invasiveness was studied using the Boyden chamber assay. Gene expression was examined by Illumina DNA microarray analysis and by Taq-Man real time RT-PCR assays. Results: E2 strongly suppressed invasiveness of ER+ breast cancer cells at concentrations below 0.01nM. At low (&amp;lt; 1 nM) concentrations, progesterone, R5020 and medroxyprogesterone acetate completely abrogated the inhibition of invasiveness by E2. It was only in a higher (5nM - 50 nM) concentration range that progestins induced invasiveness in the absence of E2. The ability of progestins to rescue invasiveness from E2 regulation was exclusively mediated by PR-A whereas PR-B mediated the E2-independent component of progestin-induced invasiveness. Overexpression of PR-A in PR-A+/PR-B+ cells lowered the progestin concentration needed to completely rescue invasiveness (to &amp;lt; 0.2 nM). Progesterone opposed E2 regulation of a distinctive set of genes, exclusively through PR-A. Conclusion: At relatively low concentrations, progesterone influences breast cancer cell invasiveness by rescuing it from E2 regulation via PR-A, whereas at higher concentrations the hormone also induces invasiveness independent of E2 signaling, through PR-B. The findings establish a direct functional link between PR-A and progression of luminal breast cancer in the context of the entire range of pre- and post-menopausal plasma and breast tissue estrogen and progesterone levels. Citation Format: Thomas B. McFall, Mugdha Patki, Rayna Rosati, Manohar Ratnam. Role of the short isoform of the progesterone receptor in hormone-regulated invasiveness of breast cancer cells at pre- and post-menopausal levels of estrogen and progesterone. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5038. doi:10.1158/1538-7445.AM2015-5038

Postmenopausal plasma sex hormone levels and breast cancer risk over 20 years of follow-up

Plasma estrogen and androgen levels are positively associated with postmenopausal breast cancer risk, but how long a single blood measurement can predict risk and whether the associations vary by tumor hormone receptor status remain unclear. We conducted nested case-control analyses within the Nurses' Health Study. Blood samples were collected in 1989-1990 and again in 2000-2002. Among postmenopausal women not using postmenopausal hormones at blood collection, 707 cases were diagnosed through June 2010, with two matched controls per case. We used unconditional logistic regression analyses to estimate the relative risks controlling for other breast cancer risk factors. The intra-class correlation coefficients for two blood measurements collected 10years apart ranged from 0.54 (dehydroepiandrosterone sulfate, DHEAS) to 0.74 (sex hormone-binding globulin, SHBG). Overall, women in the top (vs. bottom) 25% of levels of estradiol, free estradiol, testosterone, free testosterone, and DHEAS were at a 50-110% higher risk of breast cancer (p (trend)<0.001). SHBG was inversely associated with risk (p (trend)=0.004). RRs were similar when comparing cases diagnosed 1-10 versus 11-20years (or 16-20years) after blood collection (p (interaction)>0.2). Except for DHEAS, the associations varied significantly by hormone receptor status (p (heterogeneity)≤0.02). For example, the RRs (95% CIs) comparing the highest versus lowest quartile were 2.8 (2.0-4.0; p (trend)<0.001) for ER+/PR+tumors versus 1.1 (0.6-2.1; p (trend)=0.98) for ER-/PR- tumors for estradiol, and 1.8 (1.3-2.5; p (trend)<0.001) versus 0.6 (0.3-1.2; p (trend)=0.35) for testosterone. One measure of circulating sex hormones in postmenopausal women can predict risk of hormone receptor positive breast cancer for up to 16-20years.

Abstract B105: Postmenopausal plasma sex hormone levels and subsequent risk of breast cancer over 20 years of follow-up

Abstract Background: Although plasma estrogen and androgen levels are significantly positively associated with risk of postmenopausal breast cancer, it is unknown how long a single blood hormone measure can predict subsequent breast cancer risk as most prior studies have included less than 10 years of follow-up. If including hormone levels into current breast cancer risk prediction models improves discriminative ability, evaluating how the hormone/breast cancer association varies by years since blood collection can help determine the most relevant time period for hormone measures. In addition, whether the associations vary by tumor hormone receptor status is unclear. Methods: We conducted a prospective nested case-control analysis within the Nurses' Health Study cohort. Blood samples were collected in 1989-1990 and then in 2000-2002. Among eligible postmenopausal women not using postmenopausal hormones (PMH) at blood collection, 796 cases were diagnosed through June, 2010. Two control subjects (n=1,583) were matched to cases on age and time of blood collection. We used unconditional logistic regression analyses to estimate the relative risks (RRs, 95%CIs) by controlling for breast cancer risk factors. Results: The intra-class correlation coefficients for the two blood measures collected 10 years apart were 0.69 for estradiol, 0.71 for testosterone, 0.54 for dehydroepiandrosterone sulfate (DHEAS), and 0.74 for sex hormone-binding globulin (SHBG). Overall, women in the top 25% of levels of estradiol, free estradiol, testosterone, free testosterone, and DHEAS (compared to the lowest 25% of levels) were at a 50-107% higher risk of breast cancer (all p-trends&amp;lt;0.004). Relative risks were similar when comparing cases diagnosed 1-10 vs. 11-20 years (and 16-20 years) after blood collection (all p-values for interaction by follow-up period&amp;gt;0.15). For example, comparing the highest with lowest (reference) quartile of hormones measured 10 year or less before breast cancer diagnosis, the multivariable RRs (95%CIs) for overall breast cancer were 2.0 (1.4-2.7; p-trend&amp;lt;0.001) for estradiol, 1.4 (1.1-1.9; p-trend=0.001) for testosterone, and 0.63 (0.47-0.85; p-trend=0.006) for SHBG. The comparable RRs (95%CIs) for blood samples collected 11-20 years before diagnosis were 2.0 (1.3-3.1; p-trend=0.002) for estradiol, 1.6 (1.0-2.4; p-trend=0.11) for testosterone, and 0.68 (0.44-1.04; p-trend=0.04) for SHBG. Importantly, with the exception of DHEAS, the associations varied significantly by hormone receptor status of the tumor. For example, the multivariable RRs (95%CIs) comparing the highest vs. lowest quartile were 2.8 (2.0-4.0; p-trend&amp;lt;0.001) for ER+/PR+ tumors (n=347) vs. 1.1 (0.6-2.1; p-trend=0.98) for ER-/PR- tumors (n=80) for estradiol (p-heterogeneity&amp;lt;0.01), 1.8 (1.3-2.5; p-trend&amp;lt;0.001) vs. 0.6 (0.3-1.2; p-trend=0.35) for testosterone (p-heterogeneity&amp;lt;0.01), and 0.46 (0.31-0.67; p-trend=0.01) vs. 1.07 (0.56-2.06; p-trend=0.67) for SHBG (p-heterogeneity=0.01). Conclusions: A single blood sex hormone measurement in postmenopausal women can predict risk of hormone receptor positive breast cancer for up to 20 years. Citation Format: Xuehong Zhang, Shelley S. Tworoger, A. Heather Eliassen, Susan E. Hankinson. Postmenopausal plasma sex hormone levels and subsequent risk of breast cancer over 20 years of follow up. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B105.

Effects of menopause and postmenopausal tibolone treatment on plasma TNFα, IL-4, IL-10, IL-12 cytokine pattern and some bone turnover markers

Objective To asses plasma TNFα, IL-4, IL-10, IL-12; urinay hydroxyproline (Hyp) and calcium as bone resorption markers in healthy postmenopausal women compared with premenopausal ones, and to observe the effect of tibolone (2.5 mg/day for 6 months) upon above mentioned parameters. Patients and methods The study involved 24 healthy postmenopausal women (study group) and 25 premenopausal women (control group). Plasma TNFα, IL-4, IL-10 and IL-12 concentrations were measured using ELISA kits. Fasting urinary Hyp was measured by Ehrlich's spectrophotometric reaction. Calcium was determined by oxalate precipitation and the redox titration procedure. Statistical significance was analysed by the Mann–Whitney U-test for unpaired data, Wilcoxon Signed Rank test for paired data, and Pearson correlation test. Results Postmenopausal women (both before and after tibolone treatment) have increased ( p < 0.001) plasma TNFα, IL-4, IL-10, IL-12; and increased urinary Hyp ( p < 0.05) and calcium ( p < 0.001) concentrations in comparison with premenopausal individuals. Tibolone treatment has no effect on reversing the increased postmenopausal plasma TNFα, IL-4, IL-10 and IL-12, but decreases urinary Hyp and calcium to premenopausal levels. There is a weak positive correlation ( r = 0.532; p < 0.05) between TNFα and IL-4 levels in postmenopausal women. Conclusions The results show that plasma TNFα, IL-4, IL-10, IL-12, urinary Hyp and calcium increase with menopause. The increase of anti-inflammatory IL-10, IL-12 and especially IL-4 is probably a compensatory mechanism, by which these cytokines conteract to pro-inflammatory TNFα, and thus balance its osteoclast activating and oxidative stress inducing effects. Tibolone is successful in decreasing of bone resorption markers (urinary Hyp and calcium), but has no effect on reversing the impact of menopause on plasma TNFα, as well as IL-4, IL-10, IL-12. The last effect probably is related with progestogenic and androgenic properties of tibolone. Clearly, further studies are necessary to establish a risk/benefit ratio for tibolone in view of its effects on bone turnover as a new hormone replacement rationale.

Quantitative trace analysis of estriol in human plasma by negative ion chemical ionization gas chromatography–mass spectrometry using a deuterated internal standard

A stable isotope dilution gas chromatography–mass spectrometry (GC–MS) assay for the trace level determination of estriol in human plasma is described. Negative ion chemical ionization (NICI) MS is used for highly specific detection. The method involves derivatization of the phenolic hydroxyl to the pentafluorobenzyl ether derivative and subsequent reaction of the remaining hydroxyls with heptafluorobutyric anhydride. This derivative allows detection of the strikingly abundant phenolate ion under NICI conditions. [2,4,17β]- 2H 3-labeled estriol was used as an internal standard. For high-level measurements (>313 ng/l) plasma was directly derivatized by extractive alkylation followed by heptafluorobutylation prior to analysis. A rapid and simple sample work up procedure was elaborated for trace level determinations (>5 ng/l plasma) using solid-phase extraction on C 18 with an absolute recovery of 92.9%. For low-level measurements, the calibration curve was linear in the range of 5 to 625 ng/l ( r=0.99993). Inter-assay analytical precisions (RSDs) were 1.29, 2.30 and 2.89% at 39, 156 and 650 ng/l plasma, respectively. For high-level measurements, calibration curve linearity was observed in the range of 0.313 to 20 μg/l ( r=0.99998). Inter-assay analytical precisions (RSDs) were 5.17, 1.92, 2.57 and 2.74% at 0.313, 0.625, 2.5 and 10 μg/l plasma, respectively. Postmenopausal plasma was used for spiked plasma samples. Sensitivity and specificity of the presented method allows adequate determination of estriol in human plasma samples.

Focus on breast cancer.

We would like to acknowledge Fredi Rojo for his assistance in the preparation of the figures.

Ethnic differences in post-menopausal plasma oestrogen levels: high oestrone levels in Japanese-American women despite low weight.

Breast cancer incidence in Japanese-American women is approaching that of US Whites. We investigated whether this shift is paralleled by similar post-menopausal plasma hormone levels in the two ethnic groups. We also included African-American and Latina women to further our understanding of possible ethnic differences in oestrogen metabolism. We measured androstenedione (A), oestrone (E1) and oestradiol (E2) in 30 Japanese-American, 39 non-Latina White (‘White’), 66 African-American and 58 Latina women. The (age-adjusted) geometric mean E1 levels were 34 pg ml−1in Japanese-Americans, 28 pg ml−1in Whites, 35 pg ml−1in African-Americans and 31 pg ml−1in Latinas. After adjustment for body mass index, Japanese-Americans had the highest mean E1 value of all groups and this was statistically significantly greater than the value for Whites (Pt-test= 0.05). The geometric mean A concentrations were also highest in Japanese-Americans. There was little ethnic difference in E2 levels. In conclusion, post-menopausal plasma oestrogen levels in Japanese-American women are at least as high as those in Whites. © 2000 Cancer Research Campaign

Oestradiol and sex hormone-binding globulin in premenopausal and post-menopausal meat-eaters, vegetarians and vegans.

Endogenous oestradiol is strongly associated with breast cancer risk but its determinants are poorly understood. To test the hypothesis that vegetarians have lower plasma oestradiol and higher sex hormone-binding globulin (SHBG) than meat-eaters we assayed samples from 640 premenopausal women (153 meat-eaters, 382 vegetarians, 105 vegans) and 457 post-menopausal women (223 meat-eaters, 196 vegetarians, 38 vegans). Vegetarians and vegans had lower mean body mass indices (BMI) and lower plasma cholesterol concentrations than meat-eaters, but there were no statistically significant differences between meat-eaters, vegetarians and vegans in pre- or post-menopausal plasma concentrations of oestradiol or SHBG. Before adjusting for BMI there were small differences in the direction expected, with the vegetarians and vegans having higher SHBG and lower oestradiol (more noticeable amongst post-menopausal women) than the meat-eaters. These small differences were essentially eliminated by adjusting for BMI. Thus this study implies that the relatively low BMI of vegetarians and vegans does cause small changes in SHBG and in post-menopausal oestradiol, but that the composition of vegetarian diets may not have any additional effects on these hormones. © 1999 Cancer Research Campaign

Biological and immunological characterization of inhibin forms in human plasma.

A number of immunoassay methods have been developed recently to detect specifically the bioactive alpha-beta A subunit inhibin dimer (inhibin A) in human plasma. However, the specificity of these assays in terms of their ability to detect the range of inhibin forms found in plasma and their relationship to bioactivity have not been investigated. Inhibin was fractionated from human follicular fluid (hFF) and serum/plasma from women stimulated with gonadotropins (IVF serum), and from postmenopausal and male plasma, using a combined immunoaffinity/preparative SDS-PAGE procedure. The molecular weight profile of inhibin was established by inhibin in vitro bioassay, three alpha-beta A subunit specific immunoassays, and three alpha subunit-directed immunoassays that detect the alpha subunit as well as inhibin A and B forms. In hFF inhibin forms of 33, 36, 55 and 66K were detected by in vitro bioassay and by most immunoassays except for 33 k inhibin, which was nondetectable by one alpha-beta A ELISA. The alpha subunit-directed assays also detected activity in the 29-31K region, in some assays in considerably high levels. In IVF serum in vitro bioactivity and immunoactivities were detected between 27 and 100K with the alpha-beta A assays failing to detect all bioactive forms. Alpha subunit-directed assays gave similar immunoactive profiles. Neither in vitro bioassay nor alpha-beta A assays detected activity in post-menopausal plasma or male plasma, while alpha subunit-directed assays showed peaks predominantly at 36 k, although at low levels. It is concluded that dimeric inhibin A specific assays detected bioactive inhibin forms in hFF and to a lesser extent in IVF serum. Alpha subunit-directed assays correlated poorly with in vitro bioassay in hFF because of the high alpha subunit levels in this sample. The higher correlation between these assays in IVF serum suggested that there was little free alpha subunit. The 36K form in male plasma may be free alpha subunit or inhibin B.

Postmenopausal hormone replacement therapy and plasma lipoproteins

Postmenopausal hormone replacement therapy and plasma lipoproteins

Alcohol, Height, and Adiposity in Relation to Estrogen and Prolactin Levels in Postmenopausal Women

Alcohol use, height, and postmenopausal adiposity have each been positively associated with postmenopausal breast cancer risk in most epidemiologic studies. The mechanism underlying these associations is unclear, although an effect of these factors on hormone levels has been hypothesized. Few previous studies have evaluated the relationship of either alcohol consumption or height with plasma hormone levels. A positive association between adiposity and plasma estrogen levels in postmenopausal women has been reported consistently. Using archived frozen plasma samples and corresponding data from participants in the Nurses' Health Study, we determined plasma hormone levels and assessed these levels in relation to alcohol consumption, height, and adiposity among postmenopausal women. Blood samples were collected from a subset of participants in the Nurses' Health Study in 1989 and 1990, then stored in liquid nitrogen. Hormone concentrations in 217 archived plasma samples (from healthy postmenopausal women) were analyzed in 1993. Spearman correlation coefficients were calculated to assess the linear association between alcohol consumption during the previous year (mean daily intake in grams per day ascertained from semiquantitative food-frequency questionnaires completed in 1990 or 1991), height, and adiposity (as measured by body mass index [BMI] in kg/m2, with weight reported at time of blood collection), and plasma hormone levels. Two-sided P values were also calculated. After controlling for age, height, smoking status, and BMI, alcohol consumption was positively associated with estrone sulfate concentrations (r = .17; P = .02); no statistically significant association was noted for the other plasma hormones measured. Mean plasma estrone sulfate levels were 159 pg/mL in women who reported no alcohol use versus 211 pg/mL in women consuming 30 g or more of alcohol per day. After adjusting for the other covariates, we observed a strong positive correlation between BMI and plasma estrogens (r ranging from .37 for estrone and estrone sulfate to .63 for bioavailable estradiol, with all P values < or = .01; prolactin was the only hormone unassociated with BMI, r = -.01). Height was unrelated to either plasma estrogens or prolactin. BMI and alcohol use were positively associated with postmenopausal plasma estrogen and estrone sulfate levels, respectively. The association of alcohol consumption and postmenopausal obesity with subsequent breast cancer risk might be mediated, at least in part, through an influence on postmenopausal plasma estrogen levels. Additional studies are needed to further quantify the relationship between alcohol consumption and plasma hormone levels and to elucidate the physiologic basis for this association.

Qualitative and quantitative differences in the isoelectrofocusing profile of biologically active lutropin in the blood of normally menstruating and post-menopausal women.

Abstract. A single bolus of 100 μg of gonadoliberin (LRH) was administered intravenously to 8 post-menopausal and 9 normally menstruating women and blood was withdrawn before and 30, 60, 120 and 240 min after LRH stimulation. The plasma samples obtained at different time intervals from women showing a sufficiently high response to LRH (menopausal: 8, menstruating: 3) were combined and 2 ml samples of each pool were fractionated in triplicate by electrofocusing on sucrose density gradient. In addition, two plasma pools, obtained 30 min following LRH stimulation, one from 4 normally menstruating women (exhibiting a relatively low LH-response) and the other from 2 normally menstruating women aged 40, were analyzed in the same way in duplicate electrofocusing experiments. The hLH activity was determined in each electrofocusing fraction by an in vitro bioassay method following elution and purification by gel filtration. The LH activity was distributed as four major peaks at pI values of 7.10 ± 0.05, 7.58 ± 0.06, 8.10 ± 0.04 and 8.54 ± 0.05 and a broad area of activity comprising a number of peaks in the pH range of 8.69–9.50. The analysis of the data revealed marked differences in the relative distribution of the various molecular species present in the blood of menopausal women and of normally menstruating women. A molecular species exhibiting a pI value of 7.10 was invariably present (10 – 15% of the total) in all samples of post-menopausal plasma (PMP) but was consistently absent from all samples of midcycle plasma (MCP). The amount of relatively 'less alkaline' material (eluted from pH range 7.37–8.32) was significantly higher (P &lt; 0.001) in the PMP samples compared to MCP samples. On the other hand, in the MCP samples the amount of relatively 'more alkaline' material eluted from the pH range 8.33–9.50 was significantly (P &lt; 0.001) higher (about 60% of the total recovered activity) compared to the PMP samples (about 30% of the total). Following LRH stimulation significant temporal changes were observed in the relative contribution of various molecular species to the hLH profile. A gradual increase, up to 60 min, in the material eluted in the pH range 6.87–7.36 in the post-menopausal plasma samples was accompanied by a gradual decrease in the material eluted in the pH range 7.84–8.32. Two hours after LRH stimulation a significant drop was found in the material collected from pH range 8.33–8.68, with a concomitant rise in the material eluted in the pH range 8.69–9.50. This last mentioned shift was also observed in the plasma of normally menstruating women. It is concluded that major differences exist in the composition of biologically active hLH species present in the peripheral blood of post-menopausal and normally menstruating women. Moreover, significant temporal changes occur in the composition of circulating hLH species following stimulation by LRH both in post-menopausal and in normally menstruating women.

A radioimmunoassay for equilin in post-menopausal plasma: Plasma levels of equilin determined after oral administration of conjugated equine oestrogens (Premarin)

Preparations containing equilin are widely used in hormone therapy at the menopause. No suitable routine assay exists for this steroid. Antisera have been raised to equilin-3-hemisuccinyl-bovine serum albumin, and have been found to be highly specific for equilin. A direct, sensitive assay for this steroid in post-menopausal plasma has been established. The implications of the cross-reaction data for the synthesis of oestrogen immunogens are discussed. Equilin has been assayed in samples obtained from oophorectomised women receiving conjugated equine oestrogens (Premarin).

Biological and immunological characterization of human luteinizing hormone: I. Biological profile in pituitary and plasma samples after electrofocusing

Pituitary and plasma pools from postmenopausal women and plasma pools from women at midcycle were fractionated by electrofocusing in sucrose density gradients. The biological LH activity was determined in each of the electrofocusing fractions by the use of an in vitro bioassay method. A heterogeneous profile of LH activity was found in both pituitary and plasma samples with a large proportion present within the pH range 6.5–10. In a total of 11 electrofocusing runs 7 main regions of high LH activity were found within this range with mean pI values (±SD) of 6.75 ± 0.08 ( n = 6), 7.33 ± 0.08 (11), 7.80 ± 0.09 (11), 8.23 ± 0.10 (11), 8.81 ± 0.04 (7), 9.17 ± 0.05 (6) and 9.55 (2). A significantly higher proportion of LH activity was found in the midcycle plasma samples (36%) in the pH regions with mean pI values of 8.81, 9.17 and 9.55 than in postmenopausal plasma (7%) and pituitary extracts (5%). This indicates that the profile of biologically active LH in women in the fertile age is different from that present in postmenopausal women. By detailed fractionation based on narrow pH range studies and the refocusing of specific peak fractions it was shown that each of the regions studied consisted of several peaks of LH activity indicating the presence of a large number of molecular species exhibiting varying degrees of LH activity. The relative proportions of these species showed considerable differences between sources but also between samples from the same source,

BIOLOGICALLY ACTIVE LUTEINIZING HORMONE (LH) IN PLASMA

ABSTRACT A recently described in vitro bioassay method for the measurement of biologically active LH (Van Damme et al. 1974) has been applied to the plasma of normally menstruating and post-menopausal women. The specificity of the procedure was established according to the following evidence: 1. Parallelism was observed between dose response curves obtained with serial dilutions of a standard LH preparation (HMG 2nd IRP) and plasma pools collected during the follicular phase, at the LH-peak, during the luteal phase and after menopause. 2. There was no evidence for the presence of any synergistic or antagonistic factor in the various plasma specimens. The assay design used to establish this consisted of assaying the standard and plasma pool separately and then together as a mixture followed by an asssessment of the difference (if any) in the potencies obtained. Strict additivity should yield a relative potency of 1.0. Plasma pools which were obtained every 2–3 days throughout the menstrual cycle were assayed using this design against the standard (HMG 2nd IRP) and against a mid-cycle plasma pool obtained at the time of the LH-peak. The latter was also assayed against partially purified plasma fractions obtained from a post-menopausal plasma pool after gel filtration and isoelectric focusing. With the exception of 3 assays, in which the estimates of relative potency were 0.91, 0.94 and 0.95, respectively, in 19 assays of additivity, the fiducial limits always included unity. 3. Non-detectable LH levels were found in the plasma or serum of either hypophysectomized or hypopituitary hypogonadal men or women treated with oestrogen/progestogen combined pills. 4. The presence of calf or human serum in the assay medium is an essential requirement for a valid comparison of standard and unknown preparations. In their absence, non-parallel dose response curves between plasma and standard were obtained. The other established criteria of reliability (sensitivity and precision) were also examined. The method is sufficiently sensitive (3.5–8.0 mIU/ml plasma; HMG (2nd IRP) as standard) for the measurement of LH throughout the cycle. The mean index of precision (λ) in 230 multiple assays was 0.040. It is concluded that the modified bioassay yields valid and reliable estimates of LH when applied to human plasma obtained throughout the menstrual cycle and after menopause.

Separation of antibody-bound and free human follicle-stimulating and luteinizing hormones by ethanol-ammonium acetate.

ABSTRACT The use of 66% ethanol containing 6.6% ammonium acetate provided a simple and economical method for the separation of antibody-bound and free 131I-labelled hormones in incubation mixtures of low protein concentrations used in the radioimmunoassay of human pituitary FSH and LH. The accuracy of the procedure was confirmed by similar results obtained in the evaluation of radiation damage to the tracer, binding of the tracer to the antibody and the separation of the antibody-bound and free 131I-labelled hormones by the use of ethanol-ammonium acetate solution and chromato-electrophoresis. The sensitivity of the measurement of the hormones was 10 pg with a precision of ± 5%; thus allowing the determination of hormone concentrations in as little as 20 to 50 μl of the plasma samples. There was a 98.5 to 102% recovery of the hormones added to the plasma samples of pre-determined hormone concentrations. The use of highly purified antigens for radioisotopic labelling and standard as well as purified antisera provided specific measurements of FSH and LH in the plasma. Various dilutions of a post-menopausal plasma sample yielded responses parallel to those obtained for the doseresponse curves of the FSH and LH standards by logit-log plots, thus confirming the validity of the assay. The mean plasma FSH and LH levels in normal men, children and in women during the luteal phase were 2.7 and 2.6, 1.1 and 1.2, and 1.8 and 2 ng/ml, respectively, whereas the plasma of hypophysectomized subjects showed trace to detectable levels of FSH and LH.

STUDIES ON PITUITARY GONADOTROPHINS IN HUMAN PLASMA

ABSTRACT Plasma pools obtained from men and postmenopausal women were extracted by an ammonium acetate-ethanol procedure and assayed by four biological systems assumed to measure »total« gonadotrophic activity, follicle-stimulating or luteinizing hormone activity. The 2nd International Reference Preparation for Human Menopausal Gonadotrophin, NIH-FSH-S2 and NIH-LH-S3 were used as standards. All assays were valid with regard to the statistical tests performed. Postmenopausal plasma contained 17 times more total gonadotrophic activity, 15 times more FSH activity and 4–6 times more LH activity than male plasma. The FSH/LH ratio was 2–4 times higher in postmenopausal than in male plasma. When postmenopausal plasma was calculated in terms of male plasma, the biological dissimilarity of the two materials was also indicated by the indices of discrimination for combinations of assays, which were significantly different from unity for all groups measuring a relationship FSH/LH.