The IGF axis in baboon pregnancy: placental and systemic responses to feeding 70% global ad libitum diet.

Information on the influence of poor maternal nutrition on the regulation of responses to pregnancy, placental and fetal growth and development is critical to a better understanding of pregnancy physiology and pathophysiology. We determined normal changes and effects of controlled and monitored moderate nutrient restriction (NR) (global nutrient intake reduced to 70% of food consumed by mothers feeding ad libitum from 0.16 to 0.5 of gestation) in the baboon, on important hematological, biochemical, and hormonal indices of fetal growth and placental function. Serum IGF-I:IGFBP-3 ratio was lower in pregnant than control non-pregnant baboons feeding ad libitum. Serum concentrations of total and free IGF-I were decreased in NR mothers compared with controls (p<0.05). The decrease in fetal IGF-I did not reach significance (p=0.057). Serum IGF-I: IGFBP-3 ratio was decreased by NR in both mothers and fetuses. Maternal serum IGF-II was unchanged by NR. Placental IGF-I mRNA and protein abundance were similarly reduced whereas IGF-II mRNA increased in placental tissue of NR compared to control mothers. Systemic (maternal) and local (placental) IGFBP-1 and IGFBP-3 mRNA and protein abundance were unchanged by NR. Type 1 IGF receptor protein in the syncytiotrophoblast increased in NR. Type 2 IGF receptor protein was present in the stem villi core, and decreased after NR. We conclude that moderate NR in this important non-human primate model significantly disrupts the maternal and placental IGF-IGFBP axis and influences placental expression of this key system at the gene and protein level. Changes observed appear to be directed toward preserving placental growth.

A prospective study of endogenous estrogens and breast cancer in postmenopausal women.

BACKGROUND: Circumstantial evidence links endogenous estrogens to increased risk of breast cancer in women, but direct epidemiologic support is limited. In particular, only a few small prospective studies have addressed this issue. PURPOSE: Our purpose was to assess breast cancer risk in relation to circulating levels of the two major endogenous estrogens, estrone and estradiol, measured before the clinical onset of the disease. METHODS: The association between serum levels of estrogens and the risk of breast cancer was examined in a prospective cohort study of 14,291 New York City women, 35-65 years of age, who received screening for breast cancer at the time of blood sampling and who had not been diagnosed with breast cancer. During the first 5 1/2 years of study, we identified 130 breast cancers among the postmenopausal group (7063 women, 35,509 person-years). The case subjects and twice as many postmenopausal control subjects were included in a case-control study nested within the cohort. Biochemical analyses for percent free estradiol, percent estradiol bound to sex hormone-binding globulin (SHBG), total estradiol, estrone, and follicle-stimulating hormone were performed on sera that had been kept at -80 degrees C since sampling. RESULTS: For increasing quartiles of total estradiol, the odds ratio (ORs) of breast cancer, as adjusted for Quetelet index (weight in kilograms divided by the square of the height in meters), were 1.0, 0.9, 1.8, and 1.8 (P value for trend = .06); the ORs for increasing quartiles of estrone were 1.0, 2.2, 3.7, and 2.5 (P value for trend = .06). For increasing quartiles of free estradiol, defined as the fraction of estradiol that is not bound to proteins, the Quetelet index-adjusted ORs of breast cancer were 1.0, 1.4, 3.0, and 2.9 (P value for trend < .01). When we considered the percent of estradiol bound to SHBG, the Quetelet index-adjusted ORs were 1.0, 0.70, 0.40, and 0.32 (P value for trend < .01), thus suggesting a strong protective effect. These associations persisted or became even stronger when analyses were restricted to women whose samples had been drawn 2 or more years before breast cancer diagnosis. CONCLUSIONS: These data represent the first confirmation in a large prospective epidemiologic study of a link between circulating estrogens and breast cancer risk. Although estrogen levels appeared to fall within the conventional limits of normality in all women under study, those who subsequently developed breast cancer tended to show higher levels of estrone, total estradiol, and free estradiol, and a lower percent of estradiol bound to SHBG than women who remained free of cancer. IMPLICATIONS: Factors that increase endogenous estrogen production or reduce the binding of estradiol to SHBG may increase a woman's risk of developing breast cancer later in life.

Purification and properties of an esterase from human breast cyst fluid.

Levels of estradiol 17 beta-ester hydrolytic activity in the breast cyst fluid (BCF) from 25 different women with fibrocystic disease of the breast were found to vary over a wide range (0-2.4 nmol/min/mg protein for estradiol acetate). On the basis of electrophoretic mobility on agarose gels, the activity from different individuals appeared to be identical. The esterase activity from a single BCF sample was purified to near homogeneity by differential ammonium sulfate precipitation, ion-exchange, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after the final purification step, showed two bands with molecular weights of approximately 22,000 and 23,000, neither of which was immunoreactive with a rabbit antibody raised to a crude esterase-free BCF preparation. Esterase activity could be demonstrated after extraction and renaturation of the protein eluted from the Mr 22,000 band. Resolution of the gel, however, was not good enough to rule out the presence of esterase activity in the Mr 23,000 protein. High performance liquid chromatography gel exclusion chromatography indicated a molecular weight of 90,000-95,000 for the esterase activity in crude BCF and approximately 225,000 for the purified activity, suggesting the native protein to be a tetramer which aggregated during purification. Although the natural substrate of the BCF esterase is unknown, the enzyme is able to cleave a variety of esters including acetate, valerate, and stearate esters of estradiol and p-nitrophenyl hexanoate. It is completely inhibited by diisopropylflurophosphate and diethylnitrophenyl phosphate and partially inhibited by NaF and ebelactone. The substrate and inhibitor profile of the enzyme indicates that it is a "B"-type carboxylesterase and not a protease. A comparison of the properties of the BCF esterase with those of esterases from the formed elements of the blood or from plasma suggests that the BCF esterase is not of blood origin and is probably derived from the cyst itself. Physiologically inactive lipoidal estrogens have been shown to be present in many human body fluids and tissues and it is possible that these esters serve as storage forms of the active hormone in hormonally sensitive tissues where the free steroid could be regenerated by hydrolysis.

Estrogen-stimulated uptake of alpha 1-protease inhibitor and other plasma proteins by the mouse uterus.

Administration of a single low dose of estradiol to the immature female mouse resulted in a rapid increase in uterine trypsin inhibitory capacity. The increase was apparent within 1 h, reached a maximum in 3--4 h, and returned to base line after 18 h. No corresponding increases were observed in liver or heart. The properties of the uterine inhibitor were found to be essentially the same as those of plasma alpha 1-protease inhibitor (alpha 1-PI). When 125I-labeled mouse plasma alpha 1-PI was given iv to immature mice the administration of estradiol caused a specific stimulation of uterine uptake of the labeled protein that closely matched the estrogen-stimulated increase in uterine trypsin inhibitory capacity. Half-maximal stimulation of uptake occurred at a dose of 0.15 microgram estradiol/animal. After 3 h 97% of the labeled alpha 1-PI taken up by the uterus was in the soluble (105,000 x g) fraction of which 50% was in the lumen. Estradiol also stimulated the uptake of soybean trypsin inhibitor, bovine serum albumin, porcine fibrinogen, and human alpha 2-macroglobulin. Under conditions where puromycin blocked the synthesis of an estrogen-stimulated uterine-specific hydrolase, puromycin had no effect on the estrogen-stimulated uptake of either mouse alpha 1-PI or mouse albumin. These results suggest that the estrogen-stimulated uptake of plasma proteins by the uterus does not require new protein synthesis.

A new hormone-response hydrolase activity in the mouse uterus.

A new hydrolase activity has been identified by its capability of cleaving t-boc-Ala-Ala-Pro-Ala-AMC, the released 7-amino-4-methylcoumarin (AMC) being quantified fluorometrically. The activity in the 28,000 X g supernatant fraction of homogenates of weanling mouse uterus was about one fifth that of the adult mouse. The administration of estradiol to the weanling mouse caused a prompt increase in uterine hydrolase, the response being biphasic with peaks at 2 and 6 h. Stimulation was dose responsive and effectively blocked by cycloheximide and puromycin. Estrogen stimulation of hydrolase activity was also observed in the kidney (one fourth that of in the uterus), but not in the heart or liver. Progesterone and testosterone were poor stimulators, but estriol was as effective as estradiol. According to its elution profile in gel filtration, a molecular weight for the hydrolase of about 60,000 is suggested. Inhibition studies in vitro with crude enzyme preparations indicate a serine protease with a SH group essential for maximal activity. The natural substrate for the hydrolase has not been elucidated. It does not solubilize [3H]elastin, and the properties seem to eliminate plasminogen or latent collagenase as possible substrates.

Hormonally sensitive esterase activity in the mouse uterus results from uptake of plasma esterase.

Mouse uterine homogenates contain an estrogen-sensitive serine esterase (hydrolase II). In immature animals, hydrolase II levels increase rapidly after the administration of estradiol. However, stimulation of de novo synthesis in the uterus has not been demonstrated. In this communication we show that hydrolase II activity in the uterus results from uptake of an esterase from the blood. We describe the purification of this protein from mouse plasma and discuss some of its properties. It appears to be similar if not identical to a sexually dimorphic plasma esterase called albumin esterase or esterase 1 by others. Consistent with this, hydrolysis of estradiol valerate and beta-alanine nitrophenyl ester by either uterine homogenates or purified esterase 1 was blocked by heating or by incubation with diisopropyl flurophosphate or antibody to esterase 1. Levels of the plasma esterase (esterase 1) were 50% higher in adult female mice than in adult males. Levels in juvenile males were in between those in the adult males and females. No significant changes in esterase 1 plasma levels in any group were detected after 4 days of treatment with either testosterone or estradiol. Like the uptake of other plasma proteins, estrogen-stimulated uterine uptake of the plasma esterase was inhibited by prednisolone, but not by puromycin. Using immunoprecipitation of [35S]methionine-labeled esterase 1 prepared in a cell-free translation system, mRNA specific for esterase 1 was found in mouse liver, but not in mouse uterus. Although the natural substrate for esterase 1 (or hydrolase II) is not known, both the purified enzyme and uterine homogenates were able to hydrolyze the valerate ester of estradiol, but not the stearate ester. However, estradiol esters are not necessarily the natural substrates of esterase 1, as only the long chain fatty esters of estradiol are reported to occur in vivo.

alpha 1-Antitrypsin- and anchorage-independent growth of MCF-7 breast cancer cells.

alpha 1-Antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACHY) are closely related protease inhibitors, synthesized primarily by the liver, which play major roles in modulation of the inflammatory response. Previously, we had shown that MCF-7 human breast cancer cells were able to synthesize active alpha 1-AT and alpha 1-ACHY and that the synthesis of both inhibitors varied among different MCF-7 sublines. We now show that when MCF-7(ML) cells (a subline synthesizing low levels of alpha 1-AT) are grown in soft agar in medium depleted of its trypsin inhibitory capacity (i.e. alpha 1-AT-free), addition of alpha 1-AT (50 micrograms/ml) significantly reduces colony formation in both the presence and absence of estradiol (34% and 44%, respectively). Under these conditions, incubation with 10(-7) M estradiol alone increased colony formation 2- to 3-fold. Colony formation was also significantly reduced by serum leukocyte protease inhibitor, which, like alpha 1-AT, is a potent inhibitor of elastase-like enzymes. We also found that a variety of inflammatory mediators, cytokines, and steroid hormones are able to stimulate synthesis of alpha 1-AT and alpha 1-ACHY by MCF-7 cells. Stimulation by interleukin-6 (IL-6; 200 U/ml), epidermal growth factor (4 nM), and estradiol (10(-7) M) was 2- to 3-fold, whereas stimulations by tetradecanoylphorbol-13-acetate (TPA; 80 nM) and IL-1 (10 U/ml) were 2- to 5-fold and 5- to 10-fold, respectively. In each instance, protein synthesis, monitored by immunoprecipitation of 35S-labeled proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and steady state mRNA levels, monitored by Northern blot analysis with specific cDNA probes, increased to the same extent. Consistent with their ability to stimulate alpha 1-AT synthesis, TPA and IL-1 reduced colony formation in the absence of estradiol by 65% and 63%, respectively. In addition, the effects of both TPA and IL-1 could be reversed by antibody to alpha 1-AT. These results suggest that local synthesis of alpha 1-AT and possibly other protease inhibitors may be important in regulating the tumorigenic potential of MCF-7 breast cancer cells.

Estrogen-stimulated uptake of plasminogen by the mouse uterus.

Administration of a single low dose of estradiol to the immature (4- to 5-week-old) female mouse caused a rapid, uterine-specific increase in the uptake of radiolabeled plasminogen from plasma. A significant increase in uptake was detectable within 30 min and reached a maximum 2-4 h after administration of the hormone. After 4 h, a substantial amount (42%) of the newly taken up plasminogen was found in the uterine lumen. Half-maximal stimulation of uptake occurred at a dose of 0.20 microgram estradiol/animal. Estrogen stimulation of uptake was not blocked by puromycin, indicating that new protein synthesis was not required. Similar results were obtained with mouse plasma albumin. Estrogen-stimulated uptake was not blocked by indomethacin (10 micrograms/g BW, iv), but was blocked by prednisolone. Approximately 50% inhibition of the stimulation induced by 0.5 microgram estradiol in these 10-g animals was accomplished with 50 micrograms prednisolone. This study extends our initial findings on the estrogen-stimulated uptake of plasma proteins by the mouse uterus and provides a mechanism by which uterine plasminogen levels can be elevated before implantation.

Estriol conjugates in human breast cyst fluid and in serum of premenopausal women.

A sensitive method was developed for the assay of the four major estriol (E3) conjugates in human breast cyst fluid and in serum during the menstrual cycle. In the cyst fluid, estriol-3-sulfate (E3-3S) was found in each of ten samples, the concentrations ranging from 240-4310 pg/ml. Estriol-16-glucosiduronate (E3-16G) was detected in six samples at levels of 19-153 pg/ml; estriol-3-glucosiduronate (E3-3G) in five samples, 13-79 pg/ml and estriol-3-sulfate-16-glucosiduronate (E3-3S-16G) in four samples, 28-152 pg/ml. Unconjugated estriol was found in three of the ten cases (12-30 pg/ml). Serum samples obtained in the follicular and luteal phases of the cycle from eight different subjects were assayed in the same way. There was considerable variation between subjects and many values were indistinguishable from zero. But preliminary data suggest that E3-3G is the predominant E3 conjugate in the serum and E3-3S-16G is quantitatively least important. It appears that E3 conjugates in the cyst fluid are not derived from the blood directly, but are produced locally from precursors which have not been identified.

Effect of protein binding on transfer and metabolism of cortisol in perfused human placenta.

The transfer and metabolism of cortisol and cortisone and the effect of protein binding on these processes have been investigated in vitro in the perfused human placenta. The clearance of cortisol in buffer, expressed as a fraction of the antipyrine transfer rate (clearance index), was 0.50 +/- 0.05 SEM in either direction. Extensive conversion to cortisone (85%) occurred during transfer. Addition of corticosteroid-binding globulin (CBG) in amounts sufficient to bind 50% of the cortisol reduced the clearance (0.40 +/- .026) insignificantly, whereas human serum albumin (HSA) in amounts sufficient to bind 50% of the cortisol reduced the clearance to 0.28 +/- 0.012 (P less than 0.001) even though the association constant for albumin is approximately 1000-fold less. The percent of conversion to cortisone did not change significantly with protein binding. The clearance index of cortisone from a protein-free perfusate was 0.74. With CBG and albumin in the same concentrations as used in the cortisol experiments, the binding of cortisone to CBG was 23% and its clearance was 0.70; with albumin, the binding was 45% and the clearance index was 0.45. The addition of albumin and CBG to the same perfusate resulted in a cortisol clearance equal to that obtained with perfusate containing only albumin. Binding to albumin may be more significant than binding to CBG in controlling the transfer rate of cortisol to the fetus.

Bile acids in human breast cyst fluid: the identification of lithocholic acid.

Breast cyst fluid (BCF) aspirated from 12 women with fibrocystic disease of the breast and sera obtained simultaneously were analyzed for bile acids. Analysis was performed by gas-liquid chromatography of the acetoxy methyl esters of the bile acids prepared after alkaline hydrolysis of the bile salts. An internal standard served to correct for methodological losses. Low levels of bile acids were found in serum samples, precluding overt hepatobiliary complications. Deoxycholic acid (17-160 mumol/L), chenodeoxycholic acid (18-305 mumol/L), and cholic acid (3-119 mumol/L) were detected in 11 of 12 samples of BCF. In 2 cases, chosen at random, the identities of the bile acids were verified by mass spectrometry. Lithocholic acid (9-23 mumol/L), a reported cocarcinogen, was detected in 6 of the 12 samples of BCF. This is the first report of the presence of lithocholic acid in BCF with confirmation by Mass spectrometry. There was no correlation between the levels of individual bile acids and those of potassium ion, Na+/K+, estriol-3-sulfate, or 16 alpha-hydroxyandrogen sulfates that had been quantified previously in these samples. There was borderline correlation between concentrations of total bile acids and K+ (P less than 0.06) and Na+/K+ (P less than 0.07). Yet to be elucidated are the mechanism of accumulation of bile acids in BCF and whether levels of particular bile acids in BCF may serve to identify that small subset of women with fibrocystic disease at risk for developing breast cancer.

On the occurrence and transport of estriol-3-sulfate in human breast cyst fluid: the metabolic disposition of blood estriol-3-sulfate in normal women.

The high concentration of estriol-3-sulfate (E3-3S) in human breast cyst fluid has been confirmed in 14 women with multiple cysts. The concentrations of E3-3S found in individual cysts drained within a short time span from the same patient were variable, the ratios ranging from unity to 40. After the iv administration of [14C]estriol or [3H]E3-3S, only minor accumulation of either isotope was detected in the cyst fluids aspirated 6.5-30 h later. Since surprisingly small amounts of isotopes were found in blood, the metabolism of [3H]E3-3S was studied in 2 normal women. The test compound was injected iv, and blood samples were taken at intervals up to 7.5 h. In addition, total urine was collected for 3 days. The blood clearance of [3H]E3-3S was rapid, with the half-life ranging from 15-30 min. However, E3-3S was only a minor component of the urine, indicating rapid tissue extraction and metabolism rather than renal excretion for the compound. The studies indicate that E3-3S of human breast cyst fluid does not equilibrate rapidly with other body pools and that its uptake, if any, from the blood would be against a gradient.

Androsterone long chain fatty acid esters in human breast cyst fluid.

We reported previously that incubation of [3H] androsterone in homogenates of human breast tumor resulted in production of long chain fatty acid esters of androsterone (A-LCFE). To identify the individual A-LCFE, breast tumor homogenates were incubated with androsterone, then submitted to solvent extraction, Celite chromatography, high pressure liquid chromatography and OH- negative chemical ionization mass spectrometry. The (M-1)-ions of the oleate, linoleate, palmitoleate, palmitate, arachidonate, and stearate esters of androsterone were produced. The first 3 cited unsaturated esters accounted for over 90% of the total. Since fibrocystic disease of the breast is a reported risk factor for the development of breast cancer, breast cyst fluids were analyzed for A-LCFE as part of an overall program to relate endocrine profiles in cyst fluid to the incidence of cancer. Breast cyst fluids were analyzed for total A-LCFE by a method involving solvent extraction, saponification, purification of the liberated androsterone, and then quantification of the steroid by RIA. The 10 fluids analyzed contained 0.52-3.79 ng/ml fatty acid esters, measured as androsterone. In 4 of these samples, the individual A-LCFE were analyzed by mass spectrometry. As in the incubation study, the unsaturated fatty acid esters predominated. In 3 samples, palmitoleate and in 1 sample, oleate predominated. The palmitate varied from undetectable to 25% of the total. The divergent total concentrations and profiles of A-LCFE indicate potential parameters for correlations with the subsequent course of fibrocystic disease of the breast.

Relationship between the concentrations of estriol sulfate and estrone sulfate in human breast cyst fluid.

Estriol-3-sulfate (E3S) is present in human breast cyst fluid (BCF) in median levels of 8.7-10.4 nmol/L, yet is barely detectable in the serum (less than 0.034 nmol/L). The source of this huge concentration of E3S is unknown. It may accumulate from blood by active transport or be synthesized and concentrated within the cyst. Since estrone sulfate (E1S) and its possible precursor, dehydroepiandrosterone sulfate (DHEAS) are elevated in BCF, E3S may originate via 16 alpha-hydroxylation of E1S. The present study examined the correlations between the levels of DHEAS and E1S with those of E3S in BCF. The sodium and potassium ions were also quantified and related to the steroid concentrations. By linear regression analysis of log-normalized data there was a highly significant correlation between the concentrations of E1S and E3S (n = 355, r = 0.690, P less than 0.001) and between DHEAS and E3S (n = 361, r = 0.577, P less than 0.001). The BCF were classified according to their K/Na ion ratios: type 1, greater than 1.0, type II, less than 0.25, and type III, 0.25-1.0. By Student's t test, the concentrations of E3S differed between each BCF Type (P less than 0.002). This was also true for E1S and DHEAS. Type 1 cysts were associated with the highest estrogen sulfate levels and type II with the lowest levels. The possible physiological importance of this observation resides in reports that the BCF type expressing the highest steroid concentrations has been related to an aporcine-like epithelial lining of the cyst wall and a somewhat higher risk for developing breast cancer. The results suggest that E3S in BCF may originate from E1S, but alternate mechanisms are not precluded.

Reliability of measurements of total, protein-bound, and unbound estradiol in serum.

Estradiol (E2) circulates in the blood in three states: unbound (U-E2), bound to sex-hormone binding globulin (SHBG-E2), and bound to albumin. There is evidence to support the concept that only U-E2 and albumin-bound E2, are bioavailable (i.e., rapidly extracted by tissues). A case-control study nested within a large cohort of women, in which we are examining the effect of estrogens on breast cancer risk, offered the opportunity to assess the reliability of measurements of E2, the percentage of SHBG-E2, and the percentage of U-E2 based on multiple annual serum specimens. Long-term (1-2 year) reliability, as estimated by the intraclass correlation coefficient, was assessed in a subgroup of 71 premenopausal and 77 postmenopausal controls for whom two or three serum specimens were assayed. In postmenopausal women the intraclass correlation coefficient for a single measurement of total E2 was only 0.51. As for the percentage of SHBG-E2, intraclass correlation coefficients were 0.83 and 0.94, and for U-E2, 0.72 and 0.77 in the premenopausal and postmenopausal groups, respectively. These data suggest that, whereas single determinations of total E2 are insufficient to reliably estimate a woman's true mean level, a single measurement of the percentage of SHBG-E2 or U-E2 is adequate to assess bioavailability of E2 in an epidemiological study, irrespective of day of the menstrual cycle.

Endogenous estrogens and risk of breast cancer by estrogen receptor status: a prospective study in postmenopausal women.

A positive association between postmenopausal serum levels of total estradiol, percentage of free estradiol, and percentage of estradiol not bound to sex hormone-binding globulin (SHBG) and breast cancer risk was recently reported by the New York University Women's Health Study (P. Toniolo et al., J. Natl. Cancer Inst., 87: 190-197, 1995). Data from this prospective study are used to assess whether the observed associations differ according to estrogen receptor (ER) status of the tumor. Between 1985 and 1991, 7063 postmenopausal women donated blood and completed questionnaires at a large breast cancer screening clinic in New York City. Before 1991, 130 cases of first primary breast cancer were identified by active follow-up of the cohort. For each case, two controls were selected, matching the case on age at first blood donation and length of storage of specimens. Biochemical analyses were performed on sera that had been stored at -80 degrees since sampling. ER information was abstracted from pathology reports. Separate statistical analyses were conducted of ER-positive, ER-negative, and ER-unknown groups (53, 23, and 54 matched sets, respectively). In each of the 3 groups, the mean estradiol and the mean percentage of free estradiol were greater (21-28% and 6-7%, respectively) in cases than in controls. Conversely, the mean percentage of estradiol bound to SHBG was 9-12% lower in cases than in controls. The logistic regression coefficients measuring the strength of the association between estradiol and its free and SHBG-bound fractions and breast cancer risk were similar in the ER-positive, ER-negative, and ER-unknown groups. These data suggest that in postmenopausal women, the association of endogenous estrogens with breast cancer risk is independent of the ER status of the tumor. This result is more compatible with the hypothesis of a progression from ER-positive to ER negative tumors than with the hypothesis that ER status identifies two distinct types of breast cancer.

The effect of ouabain on placental transport of 86Rb.

Human placental fragments concentrate 86Rb 10--20-fold during a two-hour incubation period. Inhibition of ouabain is dose-dependent, reaching 90 + per cent at a concentration of 5 x 10(-5) M. The clearance index of 86Rb across the perfused human placenta is 0.34 +/- 0.08, comparing to previously reported indices for Na22 and Cl36 of 0.28 and 0.41, respectively. Ouabain in concentrations up to 5 x 10(-5) M had no detectable effect on transfer across the placenta. The clearance index of ouabain is low, averaging 0.07 in 3 experiments. 3H-ouabain is not detectably bound to albumin or placental homogenate.

Serum estradiol-binding profiles in postmenopausal women undergoing three common estrogen replacement therapies: associations with sex hormone-binding globulin, estradiol, and estrone levels.

OBJECTIVE: To compare the effects of three commonly prescribed estrogen replacement therapies-oral conjugated equine estrogens (CEE; n = 37), oral micronized estradiol (ME; n = 25), and transdermal estradiol (TE; n = 24)-on the binding characteristics of plasma estradiol as related to the concentrations of blood sex hormone-binding globulin (SHBG), estradiol, and estrone. DESIGN: Menopausal volunteers, opting for estrogen replacement therapy, gave blood at 0, 2, and 4 months. SHBG was assayed by automated immunoabsorbent technology. Estradiol and estrone were determined by quantitative gas chromatography/mass spectrometry. After tritiated estradiol was added to serum, the percentage of estradiol not bound to protein was determined by ultrafiltration and the percentage of estradiol bound to SHBG was measured by a method exploiting that this protein, even when bound to estradiol, binds avidly to Concanavalin A-Agarose. RESULTS: In each study, 2- and 4-month data were similar. Increases in SHBG concentrations were 100% (p < 0.001), 45% (p < 0.001), and 12% (nonsignificant) for subjects who were receiving CEE, ME, and TE regimens, respectively. Decreases in the percentage of estradiol not bound to protein and increases in the percentage of estradiol bound to SHBG correlated with changes in the concentrations of this protein mediated by the therapies. The order for increases in estradiol was ME-TE >> CEE, whereas for estrone, the order was ME > CEE >> TE, divergent from the SHBG responses. CONCLUSIONS: The diverse responses observed can be explained by differences in the estrogen load delivered to target tissues as controlled by the intermediary circulation and metabolism of the hormones introduced in these regimens.

Estradiol esters can replace 17 beta-estradiol in the stimulation of DNA and esterase synthesis by MCF-7 cells: a possible role for the estrogen-sensitive MCF-7 cell esterase.

In this communication we extend our earlier observations on estrogen-sensitive carboxyl esterases in MCF-7 human breast cancer cells able to hydrolyze esters of estradiol. Using either estradiol acetate or p-nitrophenyl hexanoate as substrates, esterase activity was found to increase 2-3-fold in MCF-7 cells maintained in the presence of 10(-8) M estradiol. Following sucrose density centrifugation, over 85% of total esterase activity was found in the cytoplasmic fraction. No esterase activity was found in spent media from growing cells. By size exclusion chromatography, estradiol acetate esterase activity exhibited a mol. wt of 45-50 kDa. Attempts to demonstrate incorporation of [3H]estradiol into estradiol fatty acid esters by the above MCF-7 cell line (203P) were unsuccessful, although, such incorporation could be demonstrated in two other MCF-7 cell sublines. Incubation of the 203P cells with 10 nM [3H]estradiol in the presence of 0.5 mM radioinert estradiol acetate resulted in the incorporation of 35 +/- 12% of the label into the estradiol acetate in 10 min. In the absence of radioinert estradiol acetate, no incorporation was observed. When MCF-7 cells were incubated with [3H]estradiol in the presence of a large excess of radioinert estradiol valerate, label was found only in estradiol valerate. Similarly, when the incubation was carried out in the presence of a mixture of radioinert estradiol acetate and valerate, label was incorporated into both esters. We conclude that the apparent formation of radiolabeled estradiol esters by MCF-7 cells incubated under the above conditions, results at least in part, from an esterase-catalyzed exchange reaction. Under conditions where no ester hydrolysis could be detected in the absence of cells, valerate and stearate esters of estradiol were found to be as effective as unesterified estradiol in stimulating esterase synthesis and the incorporation of [3H]thymidine into DNA. These results are consistent with a model in which an intracellular esterase in MCF-7 cells can generate estradiol from an exogenous lipoidal steroid and elicit an estrogen response.

On the processing of saliva samples for progesterone assay.

Reports from several laboratories indicate that the concentration of progesterone in the saliva provides a valid indicator of corpus luteum function. However, optimal conditions for the treatment and storage of saliva specimens prior to analysis was not addressed in these papers. We have found that 1) sonication of saliva in brief bursts produces a homogeneous sample from which progesterone is removed quantitatively by a single extraction with hexane in a vortex mixer, and 2) prompt freezing of saliva is important since storage of samples at room temperature for 48 hours results in a significant decrease in radioimmunoassayable progesterone. Four normal women provided daily saliva specimens throughout one menstrual cycle and serum samples every 3 days during the luteal phase. Excellent correlations between the progesterone profiles in the two fluids were obtained.