2-hydroxyestradiol and 16-hydroxyestrone

Although 17β-estradiol (E2) deficiency has been linked to the development of osteoarthritis (OA) in middle-aged women, there are few studies relating other estrogens and estrogen metabolites (EMs) to this condition. We developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method to measure the levels of six EMs (i.e., estrone, E2, estriol, 2-hydroxyestrone, 2-hydroxyestradiol, and 16a-hydroxyestrone) in healthy pre- and postmenopausal women and women with OA. This method had a precision ranging from 1.1 to 3.1% and a detection limit ranging from 10 to 15 pg. Compared to healthy women, serum-free E2 was lower in the luteal and postmenopausal phases in women with OA, and total serum E2 was lower in postmenopausal women with OA. Moreover, compared to healthy women, total serum 2-hydroxyestradiol was higher in postmenopausal women with OA and total serum 2-hydroxyestrone was lower in both the luteal and follicular phases in women with OA. In conclusion, our HPLC-ESI-MS/MS method allowed the measurement of multiple biochemical targets in a single assay, and, given its increased cost-effectiveness, simplicity, and speed relative to previous methods, this method is suitable for clinical studies.

Topics: Adult; Aged; Chromatography, High Pressure Liquid; Estradiol; Estriol; Estrogens; Estrone; Female; Follicular Phase; Humans; Hydroxyestrones; Limit of Detection; Luteal Phase; Middle Aged; Osteoarthritis; Postmenopause; Premenopause; Spectrometry, Mass, Electrospray Ionization; Statistics, Nonparametric

The main goal of our study was to assess estrogen contents of breast tumor tissues, having different estrogen receptor status, in relation to long-term follow-up of patients.. Twenty-one breast cancer cases, all collected from January 1986 to January 1988 at the M. Ascoli Cancer Hospital Centre in Palermo, were included in the study and compared with 6 healthy women as a control group. Average follow-up time of patients was 144 +/- 10 months. The estrogen receptor status of tissues was determined by both ligand binding and immunohistochemical assays. A high performance liquid chromatography-based approach, jointly with gas chromatography/mass spectrometry, was used to identify and measure main estrogens, various hydroxyestrogens, and their methoxy derivatives in both normal and tumor tissues.. Although variable concentrations of hydroxylated estrogens were detected, they consistently accounted for >80% of all of the estrogens. Significantly greater amounts of both 2- and 4-hydroxyestradiol, along with a marked increase of 16 alpha-hydroxyestrone (OHE(1)), were observed in cancer with respect to normal breast tissues. A significant positive association was observed with elevated 16 alpha OHE(1) (P = 0.015) in patients alive, leading to significantly lower (P = 0.043) 2OHE(1):16 alpha OHE(1) ratio values. Conversely, ratio values of 4:2 hydroxy+methoxy estrogens was significantly lower (P = 0.006) in deceased patients. Using cutoff values of 1.2 for 4:2 hydroxy+methoxy ratio and 150 fmol/mg tissue for 16 alpha OHE(1) we achieved a clear-cut separation of patients, with over-cutoff patients having 147 months and under cutoff patients showing only 47 months median survival time (P = 0.00008).. Our data imply that individual hydroxyestrogens may have a distinct role in the onset and the clinical progression of breast cancer, with greater 16 alpha OHE(1) levels being in turn associated to cancer with respect to normal tissues and to a prolonged survival of breast cancer patients.

Topics: Adult; Aged; Binding Sites; Breast Neoplasms; Chromatography, High Pressure Liquid; Estradiol; Estrogens; Estrogens, Catechol; Female; Follow-Up Studies; Humans; Hydroxyestrones; Immunoenzyme Techniques; Middle Aged; Receptors, Estrogen; Survival Rate

To constitute a method to determine the estradiol metabolites in human liver microsome in low concentration of estradiol.. Use high performance liquid chromatography after solvent extraction, evaporation, and reconstitution to separate the metabolites and use a electrochemistry detector to detect the metabolites.. With a mobile phase of acetic acid buffer-acetonitrile (50:50, v/v, pH 4.5) at flow rate of 1.0 mL/min and a potential of +0.7 V vs Ag/AgCl, all six composition were well separated and satisfactorily detected. There are E3, 16alpha-OHE1, 2-OHE2, E1, and two unidentified composition. The minimum detectable amount is about 100 p g on column. This method is sensitive enough to detect E1 in a substrate concentration of 1 micromol/L.. The method can be used to study the metabolism mechanism of estradiol in liver microsome.

Topics: Adult; Chromatography, High Pressure Liquid; Estradiol; Estriol; Estrone; Humans; Hydroxyestrones; Microsomes, Liver

The comparative mitogenic activities of 17beta-estradiol (E2) and four metabolites, 2-hydroxyestradiol (2-OHE2), 2-hydroxyestrone (2-OHE1), 16alpha-hydroxyestradiol (16alpha-OHE2) and 16alpha-hydroxyestrone (16alpha-OHE1) were determined in estrogen receptor (ER)-positive MCF-7 and T47D human breast cancer cells. E2 (1 nM) induced a 7- to 13-fold increase in cell number in both cell lines compared to untreated cells and the mitogenic potencies of 16alpha-OHE1 or 16alpha-OHE2 were comparable to or greater than E2. In contrast, 2-OHE1 and 2-OHE2 were weak mitogens in both cell lines and in cells cotreated with 1 nM E2 and 100 or 1000 nM 2-OHE1 or 2-OHE2, there was a significant inhibition of hormone-induced cell proliferation. The comparative ER agonist/antagonist activities of E2 and the metabolites on transactivation were determined in T47D cells transiently transfected with constructs containing promoter inserts from the cathepsin D (pCD) and creatine kinase B (pCKB) genes. E2, 16alpha-OHE2 and 16alpha-OHE1 induced reporter gene activity in both MCF-7 or T47D cells transfected with pCKB or pCD. In contrast, 2-OHE1 and 2-OHE2 did not exhibit ER agonist activity for these transactivation assays, but in cells cotreated with E2 plus 2-OHE1 or 2-OHE2, there was a significant decrease in the hormone-induced response. These results demonstrate that 16alpha-OHE1/16alpha-OHE2 exhibit estrogenic activities similar to that observed for E2, whereas the 2-catecholestrogens are weak ER agonists (cell proliferation) or antagonists (cell proliferation and transactivation).

Topics: Breast Neoplasms; Cathepsin D; Cell Division; Creatine Kinase; Estradiol; Estriol; Female; Humans; Hydroxyestrones; Isoenzymes; Promoter Regions, Genetic; Receptors, Estrogen; Recombinant Proteins; Transfection; Tumor Cells, Cultured

The effect of endogenous estrogens on sex-hormone-binding globulin (SHBG) production was studied in HepG2 cells. 17 beta-estradiol, estrone, and estrogens from both 2- and 16 alpha-hydroxylative pathways stimulated SHBG production, but not in parallel with their binding affinities for the estrogen receptor. Thus, the underlying mechanism may be other than a pure interaction with the estrogen receptor.

Topics: 2-Methoxyestradiol; Carcinoma, Hepatocellular; Estradiol; Estriol; Estrogens; Estrone; Humans; Hydroxyestrones; Liver Neoplasms; Receptors, Estrogen; RNA, Messenger; Sex Hormone-Binding Globulin; Tumor Cells, Cultured