The Effective Free Fraction of Estradiol and Xenoestrogens in Human Serum Measured by Whole Cell Uptake Assays: Physiology of Delivery Modifies Estrogenic Activity

Abstract

The biological activity of natural estrogens is influenced by the degree to which they bind to serum proteins. To determine directly how serum affected the uptake of estradiol, we compared the whole cell uptake of [3H]estradiol in intact MCF-7 human breast cancer cells from serum-free medium with the uptake from 100% serum from adult men. In estrogen receptor saturation assays, 28.9 times more estradiol was required in serum to occupy the same number of estrogen receptors as was required in serum-free medium (SFM), suggesting that the effective free fraction of estradiol in adult male serum was 3.46% (1/28.9). Since most xenoestrogens are not available in tritium-labeled form, the cell uptake of unlabeled xenoestrogens could not be measured directly with saturation analysis. Therefore, we developed the relative binding affinity-serum modified access (RBA-SMA) assay to determine the effect of serum on the access of nonradioactive xenoestrogens to estrogen receptors within intact MCF-7 cells. Serum modified access (SMA) was calculated by dividing the relative binding affinity (RBA, relative to estradiol) measured in 100% serum, by the RBA measured in serum-free medium. An SMA > 1 indicated that the xenoestrogen had greater access to estrogen receptors than estradiol from serum. In contrast, an SMA < 1 indicated that the xenoestrogen had less access to estrogen receptors from serum than did estradiol. The synthetic estrogen diethylstilbestrol (DES) binds poorly to sex hormone binding globulin (SHBG), and DES showed enhanced access in serum, SMA = 6.2. Additional calculations through the Ki (inhibition constant) indicated that this corresponded to an effective free fraction of 26.9% for DES in serum. The phytoestrogens, coumestrol, genistein, and equol, showed substantial enhanced access in serum, over 10-fold relative to estradiol (SMA = 12.1, 10.3, and 11.3, respectively), and effective free fractions in serum of 47.8, 45.8, and 49.7%, respectively. Since most in vitro assays of xenoestrogens do not address how serum influences their bioactivity, the estrogenic activity of these phytoestrogens would be underestimated. Conversely, biochanin A showed decreased access from serum (SMA = 0.44) and had an effective free fraction of 2.4%; its estrogenic activity would be overestimated in serum-free assays.

Tools to evaluate estrogenic potency of dietary phytoestrogens:A consensus paper from the EU Thematic Network “Phytohealth” (QLKI-2002-2453)

“…due to the high background of the fluorescein labeling in crude protein extracts (Nagel et al, 1998). On the other hand, fluorescence polarization made possible high-throughput ERbinding screening of hundreds compounds with a very favorable time-cost ratio.…”

Animal Models Impacted by Phytoestrogens in Commercial Chow: Implications for Pathways Influenced by Hormones

“…This biotransformation does not reduce the biological potency of phytoestrogens in soy protein because equol shows more than a one order of magnitude greater affinity for the estrogen receptor than its precursor in soy protein (Kuiper et al, 1998a(Kuiper et al, , 1998b. Furthermore, although estradiol is mainly bound to serum sex hormone-binding globulin and albumin, isoflavones are less avidly bound to serum proteins, making them more available for receptor occupancy (Nagel et al, 1998), which should potentially magnify their effectiveness. Overall, the mean serum concentrations of isoflavones measured in adult rodents fed the commonly used commercial rat-specific Purina 5001 diet or the 5010 mouse diet exceed the endogenous estrogen levels 30,000-to 60,000-fold.…”

Cross-species and interassay comparisons of phytoestrogen action.

“…An early study using dilute human serum reported phytoestrogen relative binding affinities of 14-27% (37), but affinities estimated in more recent studies using partially purifed SHBG range from 0.01 to 0.1% (38,39) ( Table 6). The low affinity of isoflavonoids would be expected to result in enhanced bioavailability in the presence of SHBG, and in fact the effective free fraction of COM and GEN in human serum is 45-50%, whereas only 4% of E 2 is free (40). Addition of human serum to whole-cell binding assays raises the relative binding affinity of isoflavonoids 10-fold (40), but normal serum concentrations of SHBG (0.002 mg/mL) reduce the activity of COM and GEN in the yeast estrogen screen assay by only 5-10%, which is similar to the reduction of E 2 activity (5%) (41).…”