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. 2019 Dec:195:105476.
doi: 10.1016/j.jsbmb.2019.105476. Epub 2019 Sep 24.

Simultaneous quantification of estrogens and glucocorticoids in human adipose tissue by liquid-chromatography-tandem mass spectrometry

Sofia Laforest  1 , Mélissa Pelletier  1 , Nina Denver  2 , Brigitte Poirier  3 , Sébastien Nguyen  4 , Brian R Walker  5 , Francine Durocher  6 , Natalie Z M Homer  7 , Caroline Diorio  8 , André Tchernof  1 , Ruth Andrew  9
Affiliations

Simultaneous quantification of estrogens and glucocorticoids in human adipose tissue by liquid-chromatography-tandem mass spectrometry

Sofia Laforest et al. J Steroid Biochem Mol Biol. 2019 Dec.

Abstract

The presence of estrogens, androgens and glucocorticoids as well as their receptors and steroid converting enzymes in adipose tissue has been established. Their contribution to diseases such as obesity, diabetes and hormone-dependent cancers is an active area of research. Our objective was to develop a LC-MS/MS method to quantify bioactive estrogens and glucocorticoids simultaneously in human adipose tissue. Estrogens and glucocorticoids were extracted from adipose tissue samples using solid-phase extraction. Estrogens were derivatized using 1-(2,4-dinitro-5-fluorophenyl)-4-methylpiperazine (PPZ) and methyl iodide to generate a permanently charged molecule (MPPZ). Steroids were separated and quantified by LC-MS/MS. The limit of quantitation for the steroids was between 15 and 100 pg per sample. Accuracy and precision were acceptable (<20%). Using this method, estradiol, estrone, cortisone and cortisol were quantified in adipose tissue from women with and without breast cancer. This novel assay of estrogens and glucocorticoids by LC-MS/MS coupled with derivatization allowed simultaneous quantification of a panel of steroids in human adipose tissue across the endogenous range of concentrations encountered in health and disease.

Keywords: Adipose; Cortisol; Cortisone; Derivatization; Estradiol; Estrone.

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Conflict of interest statement

AT is the recipient of research grant support from Johnson & Johnson Medical Companies and Medtronic for studies unrelated to this publication.

Figures

Fig. 1
Fig. 1
Pathways of glucocorticoid and estrogen metabolism and hypothesized cross-regulation in adipose tissue. A) Alternative splicing of the rate-limiting enzyme aromatase (CYP19A1). Tissue-specific aromatase expression in normal adipose tissue is conferred by promoter I.4 which possesses a glucocorticoid response element. B) Androstenedione and testosterone are converted into estrogens by the action of aromatase. Androstenedione and testosterone as well as estrone and estradiol are interconverted by the action of several 17β-HSDs. Cortisone is converted into active cortisol by the action of 11β-HSD type 1 (reductase) which predominates over 11β-HSD type 2 in adipose tissue. Higher concentrations of estrogens may inhibit the activity of 11β-HSD type 1. Expression of enzymes in black squares are increased in the adipose tissue as a function of adiposity.
Fig. 2
Fig. 2
Formation of estrogen derivatives.
Fig. 3
Fig. 3
Mass chromatograms of glucocorticoids and MPPZ derivatives of estrogens following analysis of an unextracted solution of standards, 1000 pg/sample. Total Ion Chromatograms and the corresponding extracted ion chromatograms showing resolution of cortisone, cortisol and derivatives of estrone and estradiol, by retention time and mass transition.
Fig. 4
Fig. 4
Mass chromatograms of glucocorticoids and MPPZ derivatives of estrogens extracted from adipose tissue. Extracted ion chromatograms at (A) the lower and (B) upper limit of quantitation and in adipose tissue (C) from control women and (D) women with breast cancer.
Fig. 5
Fig. 5
Calibration curves of glucocorticoids and MPPZ derivatives of estrogens following extraction. A) Cortisone, B) Cortisol, C) Estrone and D) Estradiol. Regression lines (representing the range covered by the standard curve) were fitted with a 1/x weighting. Grey circles represent values of patient samples falling in the linear range and black squares represent those values requiring extrapolation.
See this image and copyright information in PMC

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