[Simultaneous determination of 7 female sex hormones in essential oil by high performance liquid chromatography-tandem mass spectrometry with isotope dilution].

A reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of 7 female sex hormones (estriol, estradiol, estrone, ethinyloestradiol, dienestrol, hexestrol, diethylstilbestrol) in essential oil was developed. The sample was extracted by ethylacetate-normal hexane solution (2:98, v/v) and the extract was purified by a silica solid phase extraction-based clean-up column. Then, the analytes were separated on an ACQUITY UPLC BEH SHELD RP18 column (100 mm x 2.1 mm, 1.7 microm) in gradient elution with the mobile phases of water and acetonitrile. The separated compounds were detected with a Waters Xevo TQ MS tandem quadrupole mass spectrometer operated in negative electro-spray ionization using multiple reaction monitoring mode. Estriol-D3, estradiol-D3 and diethylstilbestrol-D6 were used as the internal standards to reduce the matrix effects. The limits of detection and quantitation for the 7 female sex hormones in essential oil were 0.3 -7 microg/kg and 1-20 microg/kg, respectively. Good linear relationships and high correlation coefficients (r2 > or = 0.997) were obtained in the mass concentration range of 20-500 microg/L. The average recoveries were 88.5%-114.8% and the intra-assay relative standard deviations were 4.8%-18.9% at the spiked levels of 20-500 microg/kg. Finally, a total of 12 samples randomly collected from different supermarkets in Zhejiang Province were screened for the 7 female sex hormones by the proposed method. The results showed that only one sample contained estradiol and estrone.

Fecal steroid monitoring for assessing gonadal and adrenal activity in the golden eagle and peregrine falcon.

We examined the efficacy of noninvasive monitoring of endocrine function via fecal steroid immunoassays in the golden eagle and peregrine falcon. High-pressure liquid chromatography analyses of fecal glucocorticoid metabolites (fGCM) revealed that minor percentages of immunoreactive fGCM co-eluted with [(3)H]corticosterone in both sexes of the eagle (2.5-2.7%) and falcon (7.5-11.9%). In contrast, most fecal estrogen metabolites in eagle and falcon females co-eluted with radiolabeled estradiol-17beta ([(3)H]; 57.6, 64.6%, respectively) or estrone ([(3)H]; 26.9, 4.1%, respectively). Most fecal progestin metabolite immunoreactivity in the female eagle (24.8%) and falcon (21.7%) co-eluted with progesterone ([(14)C]). Most fecal androgen metabolite immunoreactivity in eagle (55.8%) and falcon (63.7%) males co-eluted with testosterone ([(14)C]). Exogenous adrenocorticotropin hormone induced increased fGCM excretion above pre-treatment in both species, but only significantly (P < 0.05) in the eagle. Both species showed increased fGCM after saline administration, suggesting the detection of 'handling stress.' Both species exhibited enterohepatic and renal recirculation of administered steroids as demonstrated by biphasic and triphasic excretion patterns. Thus, noninvasive fecal hormone monitoring is a valid and promising tool for assessing gonadal and adrenal status in rare and threatened birds-of-prey.

Assessment of total effective xenoestrogen burden in adipose tissue and identification of chemicals responsible for the combined estrogenic effect.

Test systems to screen for estrogenicity and appropriate biomarkers of human exposure are required for epidemiological studies of endocrine disruption. We addressed these issues by developing and standardising a method to assess the total estrogenic xenobiotic burden in human adipose tissue. In this study, which is the continuation of a previous work, we have improved the protocol for extensive fractionation of a higher number of tissue samples in order to investigate bioaccumulated xenoestrogens that are candidates for estrogenicity and to assess their combined estrogenic effect. This was achieved by extensive HPLC separation of xenoestrogens from endogenous hormones followed by testing of individual fractions in the E-Screen test for estrogenicity. Organochlorine pesticides, PCBs and halogenated bisphenols and alkylphenols were collected in the most lipophilic fractions, followed by progestins, androgens and estradiol esters, and then by steroidal estrogens; phyto- and myco-estrogens were collected around the end of the run. These results were confirmed by exhaustive chemical analysis. In 458 human adipose tissue samples, the total effective xenoestrogen burden was positive in 75% of samples in the pooled fraction that contained organohalogenated xenoestrogens (mean 515.3 pM Eeq/g lipid; range 0-14.5 nM) and in 82% of samples in the pooled fraction where natural estrogens eluted (mean 696.6 pM Eeq/g lipid; range 0-12.9 nM). Organochlorine pesticides emerged as candidate chemicals for the estrogenicity of the first pooled fraction, because DDT and derivatives were present in 98.3% of the samples. However, no correlation was found between the concentration of any single chemical and the estrogenicity determined in the bioassay. There may be several reasons for this lack of concordance: (i) the estrogenic effects depicted in the E-Screen bioassay are a consequence of the combined effect of several organohalogens or (ii) the proliferative effect is due to other chemicals not measured. Because additive, synergistic or antagonistic mechanisms may account for the final effect observed in the pooled fractions, the approach proposed in this work is more appropriate for exposure assessment in epidemiological studies than the determination of individual chemicals in human samples.

A sensitive enzyme immunoassay (EIA) for the determination of melengestrol acetate (MGA) in adipose and muscle tissues.

The development of a sensitive screening method of MGA residues in bovine perirenal fat and muscle based on a competitive microtitration plate enzyme immunoassay is described. The samples were extracted with petroleum ether and purified with octadecyl-silica-cartridges. The detection limit for fat was 0.4 ng/g andfor muscle tissue 0.05 ng/g, much lower than requiredfor reliable detection of positive samples. The mean recovery rates of fortified samples amount to 75%, the mean intraassay variations to 7% and the interassay variation to 13%. Determination limits were validated for fat at 2 ng/g and for muscle at 0.1 ng/g. The efficiency of the new screening method was successfully demonstrated by the direct comparison to GC-MS and LC-MS methods performed at natural positive samples originating from an animal experiment in which the labelled dose (0.5 mg per animal and day) with and without a 48 h withdrawal period or 3-fold or 10-fold the amount of MGA, respectively, was fed to Holstein Frisian heifers. In conclusion, this new screening method can be used for sensitive determination of MGA residues in adipose tissues even after low treatment doses or longer withdrawal periods.

Binding activity of 5alpha-reduced gestagens to the progestin receptor from African elephant (Loxodonta africana).

Recent findings in the African elephant (Loxodonta africana) indicate that the major progestins contained within and biosynthesized by corpora lutea are 5alpha-reduced metabolites and that progesterone is quantitatively of minor importance. The specific gestagenic action within the reproductive tract of elephants was determined by measurement of relative binding affinity of the respective progestins to the gestagen receptor extracted from elephant endometrium. The cytosol was incubated with 40 nmol/liter [3H]ORG-2058 and increasing concentrations of the tested progestin. Progesterone (P4), 5alpha-pregnane-3,20-dione (DHP), and 5alpha-pregnane-3alpha-ol-20-one (5alpha-P-3OH) were used. The competition for binding sites on the progestin receptor was shown by decreasing counts measured after extraction with scintillation fluid. The progestin concentration which induced a 50% reduction of measured counts was estimated (C50) and relative binding affinity of progesterone to other progestins was calculated (RBA = C50progestin/C50p4). The relative binding affinity of DHP to P4 at the gestagen receptor of elephant endometrium was equivalent. The other 5alpha-reduced progestin (5alpha-P-3OH) showed no competition to the [3H]ORG-2058 receptor binding. We conclude that the biological significance of P4 and DHP at the receptor level is very similar. The higher quantitative levels of DHP in corpus luteum and serum support the hypothesis that this progestin is the major gestagen in the elephants, whereas 5alpha-P-3OH is an inactive metabolite.

Sexual differentiation of the human midtrimester brain.

It is firmly believed that sexual differentiation of the brain is linked with external genital differentiation in timing as an in utero event in the human. An extensive search for oestrogen, androgen and progestin receptors failed to show their presence despite adequate controls in cytosols from human fetal brain of gestational ages 14-20 weeks. It is possible that the receptors are present in levels so low that they are undetectable by present-day methods. Our results would indicate that hormonally influenced in utero brain sexual differentiation is most unlikely to occur as a mid-trimester event.

A case-control study of breast cancer in relation to the use of steroid contraceptive agents.

In a case-control study of 141 cases of breast cancer and 279 control patients from the Royal Prince Alfred and Westmead Hospitals during 1980-1982, we found similar risk factors to those reported for other populations. There was no statistically significant evidence of an increased risk of cancer from the use of oral contraceptive agents; the crude estimate of relative risk for patients who had used oral contraceptive agents at some time was 1.3 with 95% confidence limits of 0.8 and 1.9. After adjustment for other risk factors (age at first live birth, age at menarche, number of pregnancies, menopausal status, bilateral oophorectomy and years of education), the estimate of the relative risk of ever having used an oral contraceptive agent was 0.9 with 95% confidence limits of 0.6 and 1.5. Further analysis in terms of duration of use and dosage also provided no evidence of an increased risk.

Localization of a synthetic progestin in the reproductive organs of the female baboon.

The uptake and retention of a radiolabeled synthetic progestin, ORG 2058, was studied in the female reproductive system of the baboon. Four estrogen-primed baboons were injected intravenously with 2.5 micrograms/kg body weight of 3H-ORG 2058. One animal, which served as a control, received an additional injection of 2.5 mg/kg body weight of unlabeled progesterone. One hour after the injections, the animals were killed and the uterus, cervix, oviduct, vagina, and labia were removed and processed for autoradiography. The cells in the germinative layers of the stratified squamous epithelium of the cervix, vagina, and labia demonstrated nuclear localization of the label. The columnar epithelium, both surface and glandular, of the uterus and cervix sequestered the synthetic steroid; however, the nuclei of the epithelium lining the oviduct were unlabeled. The nuclei of the fibroblasts and of the smooth muscle cells were labeled in all the organs studied. These preliminary observations suggest that there is a stage in the reproductive cycle in which progesterone receptors are contained in the stromal cells of the oviduct but are absent in the epithelium.

Oestrogen receptor assay. False positive analysis?

The influence of unlabelled oestradiol, DES, testosterone and R-5020/org 2058 on tritiated oestradiol binding was investigated in 162 ER positive cases of patients with primary breast carcinoma. A dextran-coated charcoal as well as a sucrose gradient method was applied. In 122 cases only unlabelled oestradiol and DES significantly displaced the binding of labelled oestradiol. In the remaining 40 cases, oestradiol, DES, as well as testosterone and R-5020/org 2058 were able to displace the high-affinity, saturable binding of tritiated oestradiol equally. Possible explanations of this new discovery are discussed.

[Model studies on incubated hen's eggs for the detection of synthetic gestagens within the framework of residue determination]. / Modelluntersuchungen an bebrüteten Hühnereiern zum Nachweis synthetischer Gestagene im Rahmen von Rückstandsbestimmungen.

Reported in this paper are model experiments with the incubated hen's egg, since an easily practicable and sufficiently accurate biological testing procedure has long been necessary for the detection of gestagen residues, in the context of food hygiene testing. The gestagens chloromadinone acetate and megestrol acetate were identified as pure substance in concentrations as low as 1 microgram/egg. These gestagens had caused in hatched chickens significant reduction in body weight as well as conspicuous loss of feathers or growth of stubble feathers. However, the same effects were not obtainable from normethisterone acetate or norgestrel.