title: Quantifying estrogen metabolism: an evaluation of the reproducibility and validity of enzyme immunoassays for 2-hydroxyestrone and 16alpha-hydroxyestrone in urine.
abstract: Rapid and simple enzyme immunoassays (EIAs) were recently developed to measure 2-hydroxyestrone and 16alpha-hydroxyestrone in unextracted urine. The balance between these competing estrogen metabolism pathways may serve as a biomarker of breast cancer risk. Before testing these assays in epidemiologic studies, we evaluated their reproducibility, and validity relative to gas chromatography-mass spectroscopy (GC-MS). Overnight 12-hr urine collections from five midfollicular premenopausal women, five midluteal premenopausal women, and five postmenopausal women were aliquoted and stored at -70 degrees C. Two aliquots from each woman were assayed with the EIAs in a random, blinded order, monthly over 4 months and 1 year later. Reproducibility over 4 months was good for both metabolites in premenopausal women (coefficient of variation = 8-14%) and satisfactory in postmenopausal women (approximately 19%). Reproducibility over 12 months remained good in premenopausal women, but was poor in postmenopausal women, with mean readings increasing 50 to 100%. Wide variation in estrogen metabolite levels enabled a single EIA measurement to characterize individual differences among premenopausal women in midfollicular (intraclass correlation coefficient = 98-99%) and midluteal phase (85-91%). A narrower range in metabolite levels among postmenopausal women reduced discrimination (78-82%). The correlation between EIA and GC-MS measurement was excellent for both metabolites (r>0.9), except for 2-hydroxyestrone in postmenopausal women (r=0.6). Analysis of absolute agreement suggested that both EIAs were less sensitive than GC-MS, and each detected nonspecific background. The low concentration of estrogen metabolites in urine from postmenopausal women may explain the problems with reproducibility and validity in this menstrual group. Accordingly, more sensitive EIAs have been developed and are now being evaluated.
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Maryland 20892, USA. zieglerr@epndce.nci.nih.gov
authors.forename: R G
authors.initials: RG
authors.lastname: Ziegler
authors.forename: S C
authors.initials: SC
authors.lastname: Rossi
authors.forename: T R
authors.initials: TR
authors.lastname: Fears
authors.forename: H L
authors.initials: HL
authors.lastname: Bradlow
authors.forename: H
authors.initials: H
authors.lastname: Adlercreutz
authors.forename: D
authors.initials: D
authors.lastname: Sepkovic
authors.forename: P
authors.initials: P
authors.lastname: Kiuru
authors.forename: K
authors.initials: K
authors.lastname: Wahala
authors.forename: J B
authors.initials: JB
authors.lastname: Vaught
authors.forename: J L
authors.initials: JL
authors.lastname: Donaldson
authors.forename: R T
authors.initials: RT
authors.lastname: Falk
authors.forename: C M
authors.initials: CM
authors.lastname: Fillmore
authors.forename: P K
authors.initials: PK
authors.lastname: Siiteri
authors.forename: R N
authors.initials: RN
authors.lastname: Hoover
authors.forename: M H
authors.initials: MH
authors.lastname: Gail
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000032:analysis; D001943:Breast Neoplasms / Q000209:etiology / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D005260:Female; D005498:Follicular Phase / Q000652:urine; D008401:Gas Chromatography-Mass Spectrometry / Q000706:statistics & numerical data; D006801:Humans; D006894:Hydroxyestrones / Q000652:urine*; D007124:Immunoenzyme Techniques / Q000706:statistics & numerical data; D008183:Luteal Phase / Q000652:urine; D008593:Menopause / Q000652:urine; D008875:Middle Aged; D009376:Neoplasms, Hormone-Dependent / Q000209:etiology / Q000378:metabolism; D015203:Reproducibility of Results; D012307:Risk Factors
chemical_list: D015415:Biomarkers; D004967:Estrogens; D006894:Hydroxyestrones; C026702:16-hydroxyestrone; C024980:2-hydroxyestrone