Comparison of liquid chromatography-tandem mass spectrometry, RIA, and ELISA methods for measurement of urinary estrogens

Abstract

Absolute and relative concentrations of estrogens and estrogen metabolites are important for clinical decisions as well as for epidemiologic, experimental, and clinical research on hormonal carcinogenesis. RIA and ELISA are routinely used for measuring estrogen metabolites in blood and urine due to efficiency and low cost. Here, we compare absolute and ranked concentrations of estrone, estradiol, and estriol measured by indirect RIA and of 2-hydroxyestrone and 16alpha-hydroxyestrone measured by ELISA to the concentrations obtained using a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which measures 15 estrogen metabolites concurrently. We used overnight urine samples collected from control women (362 premenopausal and 168 postmenopausal) participating in a population-based case-control study of breast cancer among Asian American women ages 20 to 55 years. When comparing RIA or ELISA levels to LC-MS/MS, absolute concentrations for the five estrogen metabolites ranged from 1.6 to 2.9 and 1.4 to 11.8 times higher in premenopausal and postmenopausal women, respectively (all P < 0.0001). However, LC-MS/MS measurements were highly correlated [Spearman r (r(s)) = 0.8-0.9] with RIA and ELISA measurements in premenopausal women and moderately correlated (r(s) = 0.4-0.8) in postmenopausal women. Measurements of the 2-hydroxyestrone:16alpha-hydroxyestrone ratio, a putative biomarker of breast cancer risk, were moderately correlated in premenopausal women (r(s) = 0.6-0.7) but only weakly correlated in postmenopausal women (r(s) = 0.2). LC-MS/MS had higher intraclass correlation coefficients (> or =99.6%) and lower coefficients of variation (< or =9.4%) than ELISA (> or =97.2% and < or =14.2%) and RIA (> or =95.2% and < or =17.8%). Comparison with the LC-MS/MS method suggests that the widely used RIA and ELISA estrogen metabolite measures may be problematic, especially at low estrogen metabolite levels characteristic of postmenopausal women.

Figure 1

Geometric mean levels of urinary estrogen metabolites (EM) in pmol/mg creatinine for premenopausal, luteal phase (A); premenopausal, nonluteal phase (B), and postmenopausal women (C). EM are grouped as either parent estrogens or by hydroxylation pathway.

Figure 1

Geometric mean levels of urinary estrogen metabolites (EM) in pmol/mg creatinine for premenopausal, luteal phase (A); premenopausal, nonluteal phase (B), and postmenopausal women (C). EM are grouped as either parent estrogens or by hydroxylation pathway.