| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Other Original Articles |
Unité INSERM 439 (F.P., N.S., B.T., P.B., J.C.N., C.S.), Pathologie Moléculaire des Récepteurs Nucléaires, Montpellier 34090, France; Service d’Hormonologie du Développement et de la Reproduction (F.P., C.S.), Hôpital Lapeyronie, 34295 Montpellier, France; and Unité d’Endocrinologie Pédiatrique (F.P., C.S.), Service de Pédiatrie 1, Hôpital Arnaud de Villeneuve, 34295 Montpellier, France
Address all correspondence and requests for reprints to: Pr. Charles Sultan, INSERM U.439, Pathologie Moléculaire des Récepteurs Nucléaires, 60 rue de Navacelles, 34090 Montpellier, France. E-mail: chsultan{at}u439.montp.inserm.fr
Abstract
The evaluation of estrogenic status is necessary for many physiological and pathological conditions in pediatric as well as adult endocrinology. Because current immunoassays exclusively measure E2—and with a sensitivity that is insufficient for prepubertal children—we developed a new recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity. This assay is based on human uterine cervix carcinoma cells, HeLa cells, that do not naturally express E2 receptor. These cells were transfected with plasmids encoding the human ER
or ß, along with an estrogen-responsive promoter fused to the luciferase gene, and called HELN and HELNß for HeLa estrogen-responsive element luciferase neomycin and ß. HELN and HELNß are able to respond to estrogens and various compounds having estrogenic activity but, because of the importance of ER in the reproductive function, we chose to work with the HELN cell line. The luciferase activity we obtained was compared with an E2 standard curve specific for each serum sample and established with stripped serum. The estrogenic bioactivity was expressed in picograms of E2 equivalents, and the detection limit was < 1 pg·ml-1 E2 equivalents. The intra and interassay error was lower than 10% and 20%, respectively. We measured estrogenic bioactivity in 18 normal prepubertal boys (age = 9.7 ± 2.4 yr), 18 normal prepubertal girls (age = 9.2 ± 1.7 yr) and 18 normal pubertal girls (age = 13.6 ± 1.8 yr). The estrogenic bioactivity in the prepubertal girls was significantly higher than in the boys, i.e. 3.53 ± 2.23 pg·ml-1 vs. 1.44 ± 0.87 pg·ml-1 (P < 0.01). A significant difference was found between the pre- and pubertal girls, i.e. 3.53 ± 2.23 pg·ml-1 vs. 26.77 ± 18.32 pg·ml-1 (P < 0.01). This ultrasensitive bioassay measures total estrogenic bioactivity of serum with very high sensitivity. It has numerous potential applications in pediatric and adult endocrinology. In addition, this assay may help to evaluate excess estrogenic activity related to aromatase overexpression or contamination by environmental chemicals.
THE EVALUATION OF estrogenic status can be helpful in a wide range of clinical conditions in children as well as adults. Comparison of estrogenic status between prepubertal girls and boys would be of interest, for example, as it has been suggested that higher E2 levels in prepubertal girls may account for their earlier onset of puberty. The diagnosis and treatment of various pediatric endocrine diseases such as prepubertal gynecomastia, premature thelarche, and gonadal dysgenesis would benefit from a sensitive method of estrogen evaluation (1, 2). Recently, several reports have pointed out a significant increase in disorders of sexual differentiation related to the estrogenic (and antiandrogenic) potencies of environmental pollutants (3). The measurement of total plasmatic estrogenic bioactivity would greatly improve the evaluation of these new environmental diseases. Moreover, information on estrogenic status in adults is of great assistance in the management of various endocrine or gynecological conditions such as menstrual disorders, breast diseases, or infertility (4, 5).
Various estrogen assays have been proposed, both indirect by evaluation of peripheral estrogen action and direct assays. Estrogenic impregnation can be indirectly evaluated, for example, by uterine length at pelvic sonography. This method is widely used in clinical practice, particularly to evaluate the adolescent girl’s pubertal status, but it is also very imprecise. The direct assays include RIA and chemiluminescent immunoassay (CLIA), which exclusively measure E2. In addition, they are sensitive enough for adult serum, but their sensitivity is insufficient for prepubertal serum, with a variable mean sensitivity ranging from 5–9 pg·ml-1, whereas E2 probably exerts its biological effects at lower concentrations. Other methods such as binding (6), e-screen (7), or reporter gene assays have been developed (8, 9, 10, 11, 12). These are able to evaluate estrogens and various compounds with estrogenic activity but they have not been applied to total human serum (13). Until now, only one method based on gene reporter yeast has been used for sensitive measurement of estrogens in the serum after ether extraction (11). We consider that it would be very helpful to evaluate estrogenic bioactivity directly from a small amount of human serum to be closer to the true physiological status.
We thus developed a HeLa cell line stably transfected by ER or ß (HELN and HELNß) (14). These cells respond specifically to estrogens and different xenoestrogens. Using this cell line, we established a bioassay system for evaluation of estrogenic bioactivity in the serum. We then evaluated the estrogenic bioactivity of serum in prepubertal boys and girls and in pubertal girls.
Materials and Methods
Chemicals and materials
Culture medium and geneticin (G418) were obtained from Life Technologies, Inc. (Cergy Pontoise-France). Luciferin was synthesized by G. Auzou according to Bowie (14A ). E2, estrone (E1), estrone sulfate (E1S), estriol (E3), T, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 
5 androstenediol (5), 4 androstenedione (4), aminogluthetimide and puromycin were purchased from Sigma (St. Louis, MO). Steroids were dissolved in ethanol at 10-3 M. C18 Oasis HLB (Oasis water-wettable hydrophilic- lipophilic balanced copolymer) was bought from Waters (Saint-Quentin-Yvelines, France). All cell culture plastics were obtained from Falcon (Merck Eurolab, Strasbourg, France) except the 96-well Cellstar plates, which were obtained from Greiner Labortechnik (Poitiers, France). A MicroBeta Trilux luminometer (EG\|[amp ]\|G Wallac, Turku, Finland) was used to detect luciferase activity in intact cells. Commercial human serum was purchased from ABCys (Paris, France).
Blood collection and serum separation
Blood was collected in glass dry tubes. After centrifugation, serum was removed, transferred to glass tubes, and frozen.
Standards
Eight hundred microliters of every patient’s serum were desteroided by incubation with 30 mg of C18 Oasis HLB batch for 10 min, followed by centrifugation (13,000 rpm/min, 10 min) and supernatant recovery. A known amount of E2 was added to this stripped serum to obtain a set of E2 standards with concentrations from 10-12–0-7 M. These standard points were incubated for 4 h at 37 C to permit an equilibrium between free estrogens and estrogens bound to sex hormone-binding globulin.
HELN cells
The stably transfected cell lines were obtained from human uterine cervix carcinoma cells, HeLa cells. These cells were transfected with estrogen-responsive element (ERE) upstream of a luciferase gene reporter and with an expression plasmid of human ER or ß. These HeLa cells transfected with ERE-ß Glob-Luc-SVNeo and pSG5-Puro-hER or ß were called HELN and HELN ß for HeLa ERE luciferase neomycin ER and ß. Selection by geneticin and puromycin was made at 1 mg·ml-1 and 0.5 µg·ml-1, respectively. Luminescent and inducible clones were identified using photon-counting cameras (Argus 100 from Hamamatsu, or Night Owl from EG&G-Berthold, Bundoora, Australia) (14). Because of the importance of ER in regulating reproductive function, we began our work with HELN. In future work, however, it would be very interesting to look for the same evaluation of estrogen activity with HELN ERß (15).
HELN ER was cultured in 150-cm2 plastic flasks in DMEM without phenol red, supplemented with 5% dextran-coated, charcoal-treated FCS, 0.25 µg·ml-1 puromycin and 0.5 mg·ml-1 geneticin. Because our cells had an intrinsic aromatase activity, an aromatase inhibitor, aminogluthetimide (AG), was added to the culture medium at a concentration of 50 µM. The cells were seeded in 96-well plates (3 x 104 cells per well) in presence of DMEM without phenol red, supplemented with 3% dextran-coated, charcoal-treated FCS and 50 µM of AG, and incubated for 8 h at 37 C. Culture medium was then removed and replaced by 100-µl DMEM without phenol red but supplemented with 50 µM AG, to which 20 µl human serum in triplicate was added. All in all, for every serum sample, we had 7 standard points (0, E2 10-7, 10-8, 10-9, 10-10, 10-11, 3 x 10-12, 10-12 M). Cells were then incubated at 37 C for 16 h.
Luciferase assay in whole cell
The culture medium was removed at the end of incubation with the test compounds. Luminescent buffer (DMEM without phenol red, 3 x 10-4 M luciferin) was added at a rate of 50 µl per well. Luciferase activity was measured using a MicroBeta Trilux luminometer.
Subjects
We studied 36 normal prepubertal children (aged 6–10 yr), 18 boys and 18 girls, and 18 pubertal girls. This latter group contained premenarchal and menarchal girls. All participants had normal physical examinations, no history of significant past illness, and no known exposure to environmental contaminants. The 36 prepubertal children had no axillary or pubic hair, no breast tissue in the girls, and a testicular volume 
3 ml in the boys.
Results
HELN cell characteristics
Specificity for estrogenic compounds.
We first compared the E2 dose-response curve with those curves obtained with several androgenic products present in human serum and susceptible to interfere with the assay. E2, DHEA, DHEAS, DHT, 5, 4, and T were serially diluted in commercial human serum that had been desteroided, as described in Materials and Methods. The HELN responses are expressed in percentage of luciferase activity measured per well, and the 100% value represents the value obtained in presence of E2 10-7 M (Fig. 1A
). DHT began to increase luciferase activity only at a concentration of 10-7 M, which is much higher than physiological concentration, and reached an activity of 24% at 10-6 M. DHEA was able to increase luciferase activity from a concentration of 10-6 M, 100 times higher than physiological concentration. DHEAS did not lead to luciferase activity at physiological concentration, but only at concentrations higher than 10-4 M. The HELN probably possesses a sulfatase enzyme that allows the switch from DHEAS to DHEA; due to its binding to ER, DHEA is then able to create luciferase activity. 5 androstenediol led to detectable but very low luciferase activity, i.e. less than 5% above the luciferase background noise, at a concentration of 10-9 M, which is equivalent to its adult physiological concentration. In prepubertal serum, this steroid interference is thus expected to be even lower. These results are not surprising because previous studies have shown that 5 androstenediol, DHT, and DHEA are able to bind ER (16). Whatever the concentration of 4 androstenedione, luciferase activity was undetectable. Various estrogenic compounds were also tested, such as E1, E1S, and E3 (Fig. 1B). E1S did not lead to any luciferase activity, whereas E1 and E3 were able to increase luciferase activity with EC50 values of 2 x 10-8 M and 2 x 10-9 M, respectively. Nevertheless, these compounds would not contribute to the estrogenic activity measured in this cell bioassay because physiological concentrations of E1 vary from 3 x10-11 M for prepubertal girls to 2 x 10-10 M for pubertal girls and, except during pregnancy, E3 level is very low (<3 x 10-11 M).
|
Enzymatic activity of HELN cells.
The luciferase activity due to T led us to think that the HELN cells had an aromatase activity. To prove this hypothesis, we tested the effect of aminogluthetimide (AG), a well known aromatase inhibitor, used at various concentrations. We noticed that the luciferase activity induced by various concentrations of T was dramatically decreased in presence of AG, whereas luciferase activity due to E2 did not vary (Fig. 2). This result indicates that the HELN cells indeed had an aromatase activity. Further concentrations of AG were tested from 5–50 µM. The maximal inhibition was found at a concentration of 50 µM. Thus, even if the T concentration in prepubertal children is very low (<3 x 10-10 M), we decided to grow HELN ER cells in culture medium supplemented with 50 µM AG.
|
Because of the biochemical differences between the composition of FCS and human sera we chose to perform standard curves in stripped human serum.
Because the C18 Oasis phase was capable of retaining estrogens as well as various environmental compounds with estrogen-like activity (data not shown), and because we have limited access to human serum, desteroidation with C18 Oasis phase in batch was preferred to other methods. We first used the C18 Oasis phase according to the manufacturer’s instructions. The compounds were eluted with methanol, but after 16 h of incubation with cells the luciferase signal was totally disrupted, expressing the HELN cell death. We thus decided to work in the total human serum. This had the advantage of taking into account the specificity of the various compounds of human serum such as growth factors and binding proteins.
Standard curve
We first performed a standard curve in commercial human serum as described in Fig. 1
. Various amounts of C18 Oasis phase and various incubation times were tested to desteroid 800 µl of serum, the minimum volume of serum to perform a standard curve. The optimal desteroidation was obtained with 30 mg of C18 Oasis phase and 10 min of incubation. The luciferase activity obtained for different concentrations of E2 are shown in Fig. 3. To determine whether luciferase response was the same between different sera, we overloaded stripped patient sera with known amounts of E2. The luciferase activity/E2 concentration curve was different for each serum (Fig. 3). Interference of growth factors, which have different concentrations depending on serum sample, could explain these differences in response to the same overloads of E2.
|
). Every estrogenic bioactivity is expressed in equivalent of nanomolar concentration of E2 in standards. Accuracy
The intraassay variation was estimated by assaying the same serum fifteen times (five triplicates) in a single assay; this was performed on three different sera. The intraassay coefficients of variation (CVs) were lower than 10%, with values of 4% (7.2 ± 0.3 pg·ml-1, mean ± SD), 9% (3.2 ± 0.3 pg·ml-1), and 9% (2.2 ± 0.2 pg·ml-1). The interassay variation was determined from five consecutive assay runs. Five sera were assayed on five separate days. Variation was lower or equal to 20% with CVs of 16% (7.4 ± 1.2 pg·ml-1, mean value ± SD), 14% (5.6± 0.8 pg·ml-1), 17% (2.9 ± 0.5pg·ml-1), 18% (2.2 ± 0.4 pg·ml-1) and 20% (1.5 ± 0.3 pg·ml-1) for each serum, respectively. As one approached the detection limit, the interassay CV increased to 25% (0.8 ± 0.2 pg·ml-1).
Sensitivity
The sensitivity of the assay was defined as mean +2 SD of multiple luciferase activities induced by Oasis-stripped human serum without added E2, it was <1 pg·ml-1.
Subject data
The mean estrogenic bioactivity expressed in E2 equivalents was 1.44 ± 0.87 pg·ml-1 in normal prepubertal boys and 3.53 ± 2.23 pg·ml-1 in normal prepubertal girls (Fig. 4A). Figure 4C shows the estrogenic bioactivity in both prebubertal and pubertal girls (26.77 ± 18.32 pg·ml-1).
|
Several methods for estrogen measurement have been developed, some of which measure exclusively E2, whereas others evaluate estrogens and compounds with estrogenic activity.
Because E2 is a very small molecule, with only one binding site, it was not possible to use immunoassay by double capture. The methods of E2 measurement are thus all based on immunoassays by competition. Now, most of these immunoassays are performed directly on the serum, which is a major advantage over assays requiring extraction of steroids. The standard E2 that is in competition with the serum E2 being measured is labeled either by a radioactive compound or by nonradioactive markers, such as an enzyme or fluorescent or chemoluminescent compounds. RIA and CLIA are the classical methods for E2 measurement. The determination of serum E2 sensitivity of these two kinds of assays has improved in the past few years. RIA sensitivity is now slightly higher than that of CLIA, but because radioactivity is associated with problems of availability, stability, safety and cost, most laboratories currently use CLIA methods (17). The limit of sensitivity for CLIA is on average 9 pg·ml-1, with the lower limit of detection being 5 pg·ml-1 of serum E2. In these conditions, this limit of sensitivity is sufficient for pubertal but not for prepubertal children.
On the other hand, several methods have been developed to evaluate estrogens and estrogenic compounds (13). Some evaluate the binding affinity of estrogens and various estrogenic compounds for the ER (6). For example, one is a direct competitive ER binding assay based on recombinant human ER synthesized in Escherichia coli. The cDNA was obtained by RT-PCR from human breast cancer cells (MCF-7). The drawback of this method is that it only estimates receptor-ligand interaction. In addition, the binding affinity of the tested chemicals sometimes varies between binding assays, probably because chemical impurities interfere with the measure.
Another method studies the proliferation of MCF-7 cells (e-screen assay) (7). This method has been used to measure the estrogenicity of various compounds. It is capable of discriminating between antiestrogens and estrogens and is very sensitive (10 pM range). But this assay needs 6 or 7 d and is thus not suitable for routine use. This method may also lack precision because ER expression in MCF-7 cells is modified by growth rate and cell density. It has actually been shown that the same e-screen assay may give different results between laboratories (13).
Several assays are based on the activation of reporter genes. Among these assays, some use yeast and others use human breast cancer cells (MCF-7 cells and T47D cells) or ovarian cells (BG-1) (9, 10). The yeast assay uses yeast stably transfected with the hER and an ERE upstream of the reporter gene lacZ encoding the enzyme ß-galactosidase (18). The limit of sensitivity is 0.01 nM for E2 equivalent. This assay is not always capable of discriminating between antagonists and agonists, such as the pure antiestrogen ICI 182.780 and the partial antiestrogen tamoxifen. One hypothesis is that regions of hER that are important for activation of the transcription of estrogen-sensitive reporter genes in yeast and mammalian cells may be different (19, 20). In addition, we do not know whether the diffusion rate of various compounds through the yeast wall is the same as for human cells.
With the aim of evaluating the estrogenic and antiandrogenic potencies of different compounds, we transfected various stable human cell lines, such as MELN cells (8), PALM cells (21), or HELN and ß cells (8). The production of stable cell lines eliminates both the necessity of constantly making DNA and the variables often associated with transient transfection procedures. These stable cell lines can grow in a medium with very low amounts of charcoal-stripped serum, thus minimizing interfering background activity resulting from endogenous hormones and hormone-like substances. Contrary to the yeast assays, this kind of assay is able to differentiate between agonist and antagonist substances.
Until now, all these assays have been used exclusively to evaluate in vitro the estrogenic activity of different substances— but not to evaluate total estrogenic bioactivity in human serum.
There is only one study on estrogen determination in serum that used gene reporter in yeast (11). In this assay, yeast was stably transfected with both the human ER and a reporter plasmid containing two copies of the frog vitallogenin ERE upstream of a ß-galactosidase gene reporter. The measurement of estrogen level was made after ether extraction and the sensitivity of this method was less than 0.02 pg·ml-1. Ether most likely allows the extraction, and thus the measurement, of not only estrogens but also various estrogenic compounds, although this has not been proved. This assay needs a minimum of 48 h. As with previous studies with yeast, the diffusion of various compounds through the yeast wall is not quantified and agonist and antagonist substances cannot be differentiated.
Among the various stably transfected cell lines described previously, we chose the HELN assay to evaluate the total estrogenic bioactivity in human serum. This cell line allowed us to work exclusively with ER, whose importance in the reproductive function is now well known, and with good sensitivity (10-12 M). The incubation of stripped serum with known amounts of E2 for 4 h at 37 C resulted in an equilibrium between free estrogens and estrogens bound to sex hormone-binding globulin. Thus, the estrogenic bioactivity measured by our method was closer to the physiological state than the E2 evaluated by CLIA or the estrogen level evaluated after ether extraction in the yeast assay.
In addition, this assay had a low detection limit (<1 pg·ml-1) and was ten orders of magnitude more sensitive than the currently used E2 measurement by CLIA. It is of great interest in pediatric endocrinology to evaluate estrogenic bioactivity in prepubertal children. We demonstrated the difference between estrogenic bioactivity in prepubertal boys (1.44 ± 0.87 pg·ml-1) compared with prepubertal girls (3.53 ± 2.23 pg·ml-1) (P < 0.01). This result contradicts previous reports using immunoassay that found E2 levels in the range of 8 pg·ml-1 in both these boys and girls (22, 23, 24). Conversely, our results are in agreement with those reported by Klein in that we both found a significant difference between prepubertal boys and girls. However, using the yeast assay, Klein found estrogen levels that were very much lower than the estrogenic bioactivity that we found. Although her population of prepubertal children was very similar to ours both in terms of number and age, comparison is difficult because of the difference in model (yeast vs. mammalian cells) and the fact that we worked on total serum and not on extraction product. We also showed a clear difference between prepubertal (3.53 ± 2.23 pg·ml-1) and pubertal (26.77 ± 18.32 pg·ml-1) adolescent girls (P < 0.01).
This assay may help to better understand various physiological phenomena, such as the difference in skeletal maturation and timing of puberty between girls and boys, which may be due, in part, to the higher estrogenic bioactivity found in prepubertal girls compared with boys (11). Endocrine problems like male gynecomastia, premature thelarche or gonadal dysgenesis could be more fully explored using this assay. In the field of adult endocrinology, the physiological estrogenic status during menopause also needs to be studied.
A key advantage of this assay is that it is capable of evaluating not only the estrogens themselves, but also environmental contaminants with estrogenic activity. For the past 15 yr, increased anomalies of sexual differentiation and reproductive function in animals and humans has aroused the interest of the medical and scientific communities. Environmental estrogenic compounds have been implicated in the development of these environmental diseases, and the HELN assay could be helpful in better evaluating these diseases.
In conclusion, we developed a new recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity. The sensitivity of this method is ten orders of magnitude more sensitive than the currently used E2 measurement by CLIA. Because it is the only assay carried out on total serum, it probably best reflects physiological status. As we discussed above, this assay will provide considerable insight into both physiological and pathological processes in both pediatric and adult endocrinology. Not least, this ultrasensitive recombinant bioassay for estrogens should be of great interest for the evaluation of aromatase overexpression as well as environmental contaminants.
Footnotes
This work was supported in part by a grant from the European Community, Contract No. QLK4CT-1999-01422.
Abbreviations: AG, Aminogluthetimide; 4, 4 androstenedione; 5, 5 androstenediol; CLIA, chemiluminescent immunoassay; DHEA, dehydroepiandrosterone; CV, coefficient of variation; DHEAS, dehydroepiandrosterone sulfate; DHT, dihydrotestosterone; E1, estrone, E1S, estrone sulfate; E3, estriol; ERE, estrogen-responsive element; HELN and HELNß, HeLa cell line stably transfected by ER or ß.
Received July 11, 2001.
Accepted November 8, 2001.
References
and ß: two receptors of a kind? Curr Med Chem 7:561–576[Medline]
This article has been cited by other articles:
 |
|
 |
|
  C. Ankarberg-Lindgren and E. Norjavaara A purification step prior to commercial sensitive immunoassay is necessary to achieve clinical usefulness when quantifying serum 17 -estradiol in prepubertal children Eur. J. Endocrinol., January 1, 2008; 158(1): 117 - 124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Roy, M. Alevizaki, and I. Huhtaniemi In vitro bioassays for androgens and their diagnostic applications Hum. Reprod. Update, January 1, 2008; 14(1): 73 - 82. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Aksglaede, A. Juul, H. Leffers, N. E. Skakkebaek, and A.-M. Andersson The sensitivity of the child to sex steroids: possible impact of exogenous estrogens Hum. Reprod. Update, July 1, 2006; 12(4): 341 - 349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Wang, F. Paris, C. S. Sultan, R. X.-D. Song, L. M. Demers, B. Sundaram, J. Settlage, S. Ohorodnik, and R. J. Santen Recombinant Cell Ultrasensitive Bioassay for Measurement of Estrogens in Postmenopausal Women J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1407 - 1413. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Sonneveld, H. J. Jansen, J. A. C. Riteco, A. Brouwer, and B. van der Burg Development of Androgen- and Estrogen-Responsive Bioassays, Members of a Panel of Human Cell Line-Based Highly Selective Steroid-Responsive Bioassays Toxicol. Sci., January 1, 2005; 83(1): 136 - 148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Raivio, L. Dunkel, S. Wickman, and O. A. Janne Serum Androgen Bioactivity in Adolescence: A Longitudinal Study of Boys with Constitutional Delay of Puberty J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1188 - 1192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Guercio, M. A. Rivarola, E. Chaler, M. Maceiras, and A. Belgorosky Relationship between the Growth Hormone/Insulin-Like Growth Factor-I Axis, Insulin Sensitivity, and Adrenal Androgens in Normal Prepubertal and Pubertal Girls J. Clin. Endocrinol. Metab., March 1, 2003; 88(3): 1389 - 1393. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, 
. Various human sera were desteroided by C18 Oasis batch and overloaded with known amounts of E2. Because the dose-response curve was different for each serum, a human serum had to be compared with its own standard curve. The upper x-axis shows the E2 concentration in human serum, and the lower x-axis shows the E2 concentration in cell culture medium. The resulting luciferase activity is shown on the y-axis. Each point represents the mean of three replicates.