title: Profiling endogenous serum estrogen and estrogen-glucuronides by liquid chromatography-tandem mass spectrometry.
abstract: Estrogens, namely, 17beta-estradiol (E(2)), are conjugated to glucuronides (G), and this metabolic conversion is part of their tissular-concentration control-mechanism. This inactivation process has been observed, in addition to the liver, in several estrogen-dependent tissues and the resulting polar metabolites are detected in circulation. We developed and validated a highly sensitive and specific mass spectrometry-based method to directly measure estrogen-G serum levels. The method uses deuterated standards but does not involve enzymatic hydrolysis, a major improvement over previous techniques. Estrone (E(1)), E(1)-sulfate, E(2), the 3-G of E(1), E(2), 2-methoxy-E(1) (2-MeOE(1)) and 2-methoxy-E(2) (2-MeOE(2)), and the 17-G of E(2) were measured in serum of 19 premenopausal and 10 postmenopausal healthy women. Two extractions, solid-phase and liquid-liquid, were performed to isolate the estrogens. Estrogens were then quantified by mass spectrometry in the negative MRM ion mode using an API3200 spectrometer with a turbo ionspray source. The method selectively measured estrogen glucuronides with sensitivity > or = 5 pg/mL, accuracy 90-111%, and reproducibility (CV = 1.4-13.3%). The method is applicable between 5 and 1000 pg/mL. For the ovarian follicular phase, the major metabolite found was E(1)-3G, with E(2)-3G and 2-MeOE(1)-3G found in lesser amounts (54, 10.4, and 7.8 pg/mL, respectively) These concentrations are 2.6- to 3-fold greater than found for luteal-phase estrogens. The concentrations of E(2)-17G and 2-MeOE(2)-3G were usually less than the limit of quantification. In serum of postmenopausal women, E(1)-3G was the most abundant estrogen found (30.9 pg/mL). Our method profiles estrogens and estrogen-glucuronides and may represent a new tool to identify biomarkers in hormone-dependent diseases.
authors.affiliation: Centre Hospitalier Universitaire du Quebec Research Center, Laval University, Quebec, Canada G1V 4G2.
authors.forename: Patrick
authors.initials: P
authors.lastname: Caron
authors.forename: Etienne
authors.initials: E
authors.lastname: Audet-Walsh
authors.forename: Johanie
authors.initials: J
authors.lastname: Lépine
authors.forename: Alain
authors.initials: A
authors.lastname: Bélanger
authors.forename: Chantal
authors.initials: C
authors.lastname: Guillemette
mesh_terms: D002853:Chromatography, Liquid; D004958:Estradiol / Q000097:blood*; D005260:Female; D020719:Glucuronides / Q000097:blood*; D006801:Humans; D011786:Quality Control; D012016:Reference Values; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D020719:Glucuronides; D004958:Estradiol