Assay of ethynyloestradiol in human serum and its binding to plasma proteins

doi:10.1016/0022-4731(80)90228-9

Journal of Steroid Biochemistry

Volume 13, Issue 7, July 1980, Pages 773-779

https://doi.org/10.1016/0022-4731(80)90228-9 Get rights and content

Abstract

A radioimmunoassay is described for the estimation of both unconjugated and conjugated ethynyloestradiol in serum. Mean values for the concentration of free ethynyloestradiol were 38–87 pg/ml, 1 to 4 h after administration and by 24 h levels found in most subjects were below the sensitivity for the method (less than 25 pg/ml). The concentration of conjugated ethynyloestradiol in serum was much higher, mean values being 370–770 pg/ml, 1 to 4 h after administration decreasing slowly to mean values of 285 pg/ml at 8 h and 100 pg/ml at 24 h. The mean half-life of total (conjugated plus unconjugated) ethynyloestradiol was 3.8 h during the period 2 to 8 h after administration and 10.7 h for the period 8 to 24 h.

By equilibrium dialysis, gel-filtration on Sephadex G200 and polyacrylamide gel electrophoresis it was shown that ethynyloestradiol in plasma bound only to albumin and that no specific binding of this steroid occurred. The apparent association constant for the binding of ethynyloestradiol to human serum albumin was 1.7 × 10⁵ l/mol.

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A method for the determination of bisphenol A (BPA) in blood was investigated using high-performance liquid chromatography-electrochemical detection (HPLC-ED) with solid-phase extraction. When BPA at the concentrations of 25–100 ng/ml were added to whole blood, BPA recoveries were 26–48%. When BPA was added to water, plasma or hemolyzed red blood cells (H-RBC), BPA recoveries in water and plasma were almost similar (94%). However, the recovery in H-RBC was very low (36–46%). When BPA and plasma were added to H-RBC, the recovery was 70–85%. In authentic bovine metHb solution, BPA decreased depending on the metHb concentration, however, BPA recovery in the solution added with more than 17% plasma was higher than that in metHb only. These suggest that metHb influences the BPA recovery in whole blood. However, an accurate determination of BPA using HPLC was easily made possible by separating RBC from plasma.
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Many estrogenic chemicals found in the environment (xenoestrogens) show a lower affinity for plasma estrogen binding proteins relative to the natural estrogens such as estradiol. These binding proteins, which include alphafetoprotein in rats and mice, sex hormone binding globulin in humans, and albumin in all species, regulate estrogen uptake into tissues. Therefore, the in vivo estrogenic potency relative to estradiol of xenoestrogens that show lower binding to these serum proteins will thus be underestimated in assays that compare the potency of xenoestrogens to estradiol and do not take serum binding into account. We have examined the effects of the binding components in serum on the uptake of a number of xenoestrogens into intact MCF-7 human breast cancer cells. Since most estrogenic chemicals are not available in radiolabeled form, their uptake is determined by competition with [³H]estradiol for binding to estrogen receptors (ER) in an 18-h assay. Serum modified access (SMA) of cell uptake of xenoestrogens is calculated as the RBA in serum-free-medium ÷ the RBA in serum, and the bioactive free fraction of xenoestrogen in serum is then also calculated. We predicted the concentration of two xenoestrogens, bisphenol A and octylphenol, required to alter development of the prostate in male mouse fetuses. Whereas octylphenol was predicted to be a more potent estrogen than bisphenol A when tested in serum-free medium, our assay predicted that bisphenol A would be over 500-times more potent than octylphenol in fetal mice. The finding that administration of bisphenol A at a physiologically relevant dose predicted from our in vitro assay to pregnant mice from gestation day 11 to 17 increased adult prostate weight in male offspring relative to controls (similar to the effect of estradiol), while the same doses of octylphenol did not alter prostate development, provided support for our hypothesis.
Absence of an effect of high vitamin C dosage on the systemic availability of ethinyl estradiol in women using a combination oral contraceptive
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Previous studies in small numbers of women have suggested that the administration of gram quantities of ascorbic acid interferes with the conversion of ethinyl estradiol (EE₂) to its sulfates, leading to higher blood levels of EE₂ The possibility of such potentiation has been investigated in 37 women using a combination monophasic oral contraceptive (30 μg EE₂ and 150 μg levonorgestrel) for two consecutive cycles. Concomitant daily administration of 1 g ascorbic acid taken $12$ hour before OC intake, was randomly assigned to the first or second cycle of OC use. On the first and 15th day of OC intake, blood samples were drawn 11 times over a 12-hour interval and C_max and AUC(0–12 h) calculated. On pill days 10 and 21, only 6-hour post-intake samples were obtained. Samples were analyzed for levels of ascorbic acid, free and sulfated ethinyl estradiol (and a number of other parameters).
C_max and AUC values for EE₂ and EE₂-sulfate in cycles with and without ascorbic acid were evaluated statistically by the Grizzle model for days 1 and 15 and the ratios of day 15/day 1 for each of the substances. No effect of ascorbic acid was observed (α = 0.05, 1−β = 0.9). Only on day 15 was there a significantly lower AUC for EE₂-sulfate in the presence of ascorbic acid intake. Thus, the competition between ascorbic acid and EE₂ for sulfation does not lead to an increased systemic availability of EE₂ and is, therefore, unlikely to be of any clinical importance. Ascorbic acid can, therefore, be removed from the list of drugs interfering with the pharmaco-kinetics of ethinyl estradiol.
Rapid high-performance liquid chromatographic assay of ethynyloestradiol in rabbit plasma
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A method for the determination of ethynyloestradiol in samples of rabbit plasma containing pentobarbital and heparin, the former used as an anaesthetic and the latter as an anticoagulant, has been developed. Quantification was carried out using a reversed-phase high-performance liquid chromatographic (HPLC) method in isocratic mode at room temperature, with electrochemical detection at an applied potential of +1 V vs. Ag/AgCl. Under these conditions, the retention time for ethynyloestradiol was ca. 2.9 min, the average recovery from plasma was 74.5%, and the limit of detection was 10 pg, corresponding to a plasma concentration of 50 pg/ml using 1 ml of plasma. Natural oestrogens, oestriol, oestradiol and oestrone showed peaks that did not interfere with ethynyloestradiol, and retention times of ca. 0.8, 2.4 and 3.4 min, respectively.
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Intrasubject and intersubject variability in the metabolism of ethynyloestradiol (EE) was assessed in a cross-over randomized study of 6 women who each received 3 months treatment with 50 μg EE and 50 μg EE with 250 μg levonorgestrel (LNG). Blood samples were collected at the end of each treatment month, assayed for EE and the half-life of elimination (T_el and bioavailability (area under the serum concentration-time curve, AUC) calculated. Intrasubject variability for T_el and AUC varied markedly; the variability was random and not correlated with the formulation administered. The intrasubject variability for T_el and AUC was 31 and 17%, respectively, and intersubject variability 66 and 95%. The intersubject range of values was more than 3-fold for both T_el and AUC and the intrasubject range about 2-fold. The pharmacokinetics of EE were not influenced by LNG; mean values for T_el and AUC were 17.3 ± 5.5 h and 11.1 ± 3.8 ng/ml/h, respectively, when EE was administered alone compared with 16.4 ± 4.8 h and 12.5 ± 3.9 ng/ml/h when given with LNG. However, EE influenced the metabolism of LNG; T_el for LNG was 19.3 ± 4.2 h when administered alone and significantly higher (30.0 ± 11.2 h) when given with EE. There was no correlation between the rate of metabolism of EE and that of LNG. The intrasubject variability shown in this and other studies suggests that genetic factors are less important in intersubject variability than previously thought. Some implications of intrasubject variability are discussed.
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A pharmacodynamic and pharmacokinetic study of the Chinese No. 1 pill, a combined oral contraceptive containing 35 μg ethynyloestradiol (EE) and 600 μg norethisterone (NET), was performed in 20 women over a period of six months. Blood samples for analysis were taken during a pretreatment cycle, the first and 6th treatment cycles and post-treatment. Minor changes in carbohydrate metabolism occurred and these were particularly noticeable when the incremental areas under the serum concentration-time curves for both glucose and insulin in response to a glucose tolerance test were calculated. No changes occurred in the serum glycosylated haemoglobin levels. The serum concentrations of all the lipids measured (total cholesterol, triglycerides, LDL-C, HDL-C and apolipoproteins AI, AII and B) were significantly increased on treatment as were levels of Factor X, SHBG and caeruloplasmin whereas antithrombin III decreased. In 38 of the 40 treatment cycles, ovulation was suppressed. In one cycle serum oestradiol and progesterone levels showed a typical ovulatory pattern and in another there was evidence of follicular activity without ovulation. Serum EE concentrations showed a similar pattern in both treatment cycles showing that co-administration of NET did not affect EE metabolism. Serum NET levels were higher in the 6th than in the first treatment cycles. On comparing pharmacodynamic and pharmacokinetic parameters, the only statistically significant correlations were between the percentage change in triglycerides and SHBG and serum NET, but not EE concentrations, and between apolipoprotein AI and serum EE.

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Assay of ethynyloestradiol in human serum and its binding to plasma proteins

Abstract

Clinica chimica Acta.

Steroids

Contraception

Steroids

Steroids

J. steroid Biochem.

Plasma levels of ethynyloestradiol after administration of ethynyloestradiol or mestranol to human subjects

J. Endocr.

Radioimmunoassay of ethynyloestradiol

J. Endocr.

Metabolism of ethynyloestradiol in man

J. Endocr.