Elsevier

Steroids

Volume 75, Issues 4–5, April 2010, Pages 297-306
Steroids

Review
Analysis of estrogens in serum and plasma from postmenopausal women: Past present, and future

https://doi.org/10.1016/j.steroids.2010.01.012Get rights and content

Abstract

Previous studies have shown that the selection of women who are at high breast cancer risk for treatment with chemoprevention agents leads to an enhanced benefit/risk ratio. However, further efforts to implement this strategy will require the development of new models to predict the breast cancer risk of particular individuals. Postmenopausal women with elevated plasma or serum estrogens are at increased risk for breast cancer. Therefore, the roles of various enzymes involved in the biosynthesis of estrogens in postmenopausal women have been reviewed in detail. In addition, the potential genotoxic and/or proliferative effects of the different estrogen metabolites as risk factors in the etiology of breast cancer have been examined. Unfortunately, much of the current bioanalytical methodology employed for the analysis of plasma and serum estrogens has proved to be problematic. Major advances in risk assessment would be possible if reliable methodology were available to quantify estradiol and its major metabolites in the plasma or serum of postmenopausal women. High performance liquid chromatography (HPLC) coupled with radioimmunoassay (RIA) currently provides the most sensitive and best validated immunoassay method for the analysis of estrone and estradiol in serum samples from postmenopausal women. However, inter-individual differences in specificity observed with many other immunoassays have caused significant problems when interpreting epidemiologic studies of breast cancer. It is almost impossible to overcome the inherent assay problems involved in using RIA-based methodology, particularly for multiple estrogens. For reliable measurements of multiple estrogens in plasma or serum, it will be necessary to employ stable isotope dilution methodology in combination with liquid chromatography–tandem mass spectrometry (LC–MS/MS). Extremely high sensitivity can be obtained with pre-ionized estrogen derivatives when employed in combination with a modern triple quadrupole mass spectrometer and nanoflow LC. Using [13C6]-estrone as the internal standard it has proved possible to analyze estrone as its pre-ionized Girard T (GT) derivative in sub-fg (low amol) amounts on column. This suggests that in the future it will be possible to routinely conduct LC–MS assays of multiple estrogen metabolites in serum and plasma at even lower concentrations than the current lower limit of quantitation of 0.4 pg/mL (1.6 pmol/L). The ease with which the pre-ionization derivatization strategy can be implemented will make it possible to readily introduce high sensitivity stable isotope dilution methodology in laboratories that are currently employing LC–MS/MS methodology. This will help conserve important plasma and serum samples as it will be possible to conduct high sensitivity analyses using low sample volumes.

Introduction

17β-Estradiol (estradiol) induces tumors in animal models and in humans and elevated estrogen levels in postmenopausal women are associated with increased breast cancer risk [1]. This is thought to arise from a dual mechanism in which estradiol can act either as a hormone to stimulate aberrant cell proliferation or as the precursor to the formation of genotoxic metabolites [2]. Estrogen biosynthesis which occurs in the breast tissue of postmenopausal women is fundamentally different from that which occurs in the ovaries of premenopausal women. Unlike the ovaries, breast tissue lacks the ability to synthesize androgen precursors. Hence, estrogen production is dependent upon the availability of circulating C-19 androgen precursors and local conversion to estrogens in target tissues such as the breast. The estrogens can then be released into the circulation, which provides biomarkers of tissue estrogen biosynthesis.

Investigative studies and new therapies have significantly improved the recurrence-free and overall survival rates in breast cancer patients [3]. However, the ability to prevent breast cancer in the first place is a much more desirable goal, particularly as the world population is aging and age is an important determinant of breast cancer risk [4], [5]. This requires the selection of women who are at higher breast cancer risk for treatment with chemoprevention agents because previous studies have shown that this approach leads to an enhanced benefit/risk ratio [6], [7]. Further implementation of this strategy will require the development of new models to predict breast cancer risk of particular individuals [8]. Postmenopausal women with elevated plasma or serum estrogens are at increased risk for breast cancer [4], [9], [10], [11], [12], [13]. Unfortunately, much of the current bioanalytical methodology employed for the analysis of plasma or serum estrogens has proved to be problematic [14], [15]. Major advances in risk assessment would be possible if more reliable methodology were readily available to quantify estradiol and its major metabolites in the plasma or serum of postmenopausal women [4]. These measurements could then be coupled with other risk factors such as mammographic density [16], bone density [17], BMI [18], and single-nucleotide polymorphisms associated with breast cancer [19] to provide an improved model of breast cancer risk [6]. In addition, the availability of sensitive and specific plasma or serum estrogen assays would make it possible to more reliably assess the effects of aromatase inhibitors in postmenopausal women [20]. The present review will focus on the enzymes involved in estrogen biosynthesis, the biological effects of different estrogen metabolites, and the analysis of total (free and non-covalently bound) forms of estradiol and its metabolites in plasma and serum.

Section snippets

Enzymology of estrogen biosynthesis in postmenopausal women

The adrenal cortex is the principal source of C19 androgens, dehydroepiandrosterone (DHEA) and androstenedione (Fig. 1). Cholesterol is first metabolized to pregnenolone by cytochrome P-450 (CYP) 11A1, the side-chain cleavage enzyme [21]. Pregnenolone is then metabolized to progesterone through the action of 3β-hydroxysteroid dehydrogenase (HSD) or to 17α-hydroxy-pregnenolone by CYP17A. 17α-hydroxy-pregnenolone is further metabolized to 17α-hydroxy-progesterone by 3β-HSD.

Estradiol metabolism

Estradiol is metabolized to a 3-glucuronide by UGT1A1 [42] and to a 17β-glucuronide by UGT1A3 and 2B7 (Fig. 2) [43], [44]. It is metabolized to estradiol-3-sulfate by SULT1A1 [45], [46], SULT1E1 [46], [47], and SULT2A1 [48]. Estradiol-17β-sulfate was also observed as a minor metabolite in SULT2A1-mediated estradiol metabolism (Fig. 2). Tissue steroid sulfatase can convert estradiol-3-sulfate back to estradiol [49]. Therefore, in menopausal women, circulating estradiol-3-sulfate can serve as a

Estrone metabolism

Estrone is metabolized to a 3-glucuronide by UGT1A8 [44] and to a 3-sulfate at high concentrations by SULT1A1 and at low concentrations by SULT1E1 (Fig. 3) [74]. Estrone also undergoes extrahepatic oxidation by CYP1A1 and hepatic oxidation by CYP1A2 and CYP3A in a similar manner to estradiol to give the catechol derivatives, 2-hydroxy-estrone and 4-hydroxy-estrone (Fig. 3) [50]. In contrast, CYP1B1, metabolizes estrone to 4-hydroxy-estrone with relatively high regioselectivity [56].

Quantitative analysis of estrogens in the plasma and serum of postmenopausal women

Estradiol and its metabolites are present in plasma and serum in the free unbound form, non-covalently bound to steroid binding proteins, and as glucuronide and sulfate conjugates. Concentrations of the free (unbound) forms of plasma and serum estrogens in postmenopausal women are in the fg/mL range, which puts them below the limit of quantitation (LOQ) of routine assays [89], [90]. The LOQ is defined as the lowest concentration of an analyte in a sample that can be quantitatively determined

Future directions

The three derivatization strategies described above make it possible to quantify plasma and serum estrogens with LOQs in the low pg/mL (pmol/L) range (Table 1) [97], [114], [115], [116], [117], [120]. The availability of methodology based on these novel ionization techniques coupled with GC–MS/MS or LC–MS/MS will greatly facilitate future studies to rigorously establish the precise levels of individual estrogens that are present in the plasma of postmenopausal women.

The ESI process requires

Acknowledgements

I acknowledge the support of NIH grants UO1ES16004 and P30ES013508 and helpful discussions with Dr. Richard Santen of the University of Virginia and Dr. Trevor Penning of the University of Pennsylvania.

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