158 Endocrine Care
Endogenous Estrogen Metabolites as Biomarkers for Endometrial Cancer via a Novel Method of Liquid Chromatography-Mass Spectrometry with Hollow Fiber Liquid-Phase Microextraction
Affiliations
Key words ▶ estrogens ● ▶ 4-hydroxyestradiol ● ▶ 2-methoxyestradiol ● ▶ endometrial cancer ● ▶ LC-MS ●
received 19.06.2013 accepted 06.03.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1371865 Published online: April 10, 2014 Horm Metab Res 2015; 47: 158–164 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence L. Li, MD, PhD Department of Gynecology The Fourth Hospital of Hebei Medical University Hebei Province 050000 P. R. China Tel.: + 86/311/86095 627 Fax: + 86/311/86095 588 [email protected]
H. Zhao1, Y. Jiang2, Y. Liu2, C. Yun1, L. Li1 1 2
Department of Gynecology, The Fourth Hospital of Hebei Medical University, Hebei Province, P. R. China Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Hebei Province, P. R. China
Abstract
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Increased levels of endogenous estrogens and their metabolites are well-known risk factors of endometrial cancer. The aim of this study was to quantitatively assess the potential for estrogen metabolites to serve as biomarkers of endometrial carcinogenesis. The following estrogen metabolites were evaluated: 2-hydroxyestradiol (2-OHE2), 2-hydroxyestrone (2-OHE1), 4-hydroxyestradiol (4-OHE2), 4-hydroxyestrone (4-OHE1), 16α-hydroxyestrone (16α-OHE1), 2-methoxyestradiol (2-MeOE2), and 2-methoxyestrone (2-MeOE1). The low content of estrogen metabolites in urine makes their measurement difficult. To address this issue, we developed a rapid, sensitive, specific, and accurate liquid chromatography-mass spectrometry (LC-MS) method, with hollow fiber liquid-phase micro-extraction (HF-
Introduction
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Endometrial carcinoma is the most common malignancy of the female genital tract and is increasing in frequency [1]. The risk of endometrial carcinoma is related to a lifelong exposure to estrogen. Estrogen plays an important role in the genesis of human endometrial cancer by increasing the mitotic activity of endometrial cells. 17β-Estradiol (E2) and estrone (E1), parent estrogens in the estrogen metabolic pathways, mainly induce tumors by stimulation of epithelial cell proliferation or the formation of genotoxic catechol estrogen-quinones [2]. Catechol estrogens, natural active metabolites of estrogens, are produced by hydroxylation of estradiol and estrone at the C-2, C-4, and C-16a positions. Catechol estrogens include 2-hydroxyestradiol (2-OHE2), 2-hydroxyestrone (2-OHE1), 4-hydroxyestradiol (4-OHE2), 4-hydroxyestrone (4-OHE1), and 16α-hydroxyestrone (16α-OHE1) [2, 3]. 2-OHE2 and 2-OHE1 are methylated to 2-MeOE2 and
Zhao H et al. Endogenous Estrogen Metabolites … Horm Metab Res 2015; 47: 158–164
LPME) for an enriched pretreatment of the sample and for the simultaneous quantification of estrogens and their metabolites in the urine samples of 23 post-menopausal female endometrial cancer patients and 23 post-menopausal healthy female controls. The levels of estrogens were found to differ between the endometrial cancer patients and the controls. The level of 4-OHE2 was elevated in patients compared with the controls, while the levels of 2-MeOE1 and 2-MeOE2 were reduced in the endometrial cancer group. The results of this study indicate an imbalance of estrogen metabolites in endometrial carcinogenesis, and that the elevation of 4-OHE2 may be used as a potential biomarker for the risk assessment of estrogen-induced endometrial cancer. Supporting Information for this article is available online at http://www.thieme-connect.de/ products.
2-MeOE1 by the enzyme catechol-O-methyltransferase (COMT) [4]. Meanwhile, 4-OHE2 plays an important role in estrogen-mediated carcinogenesis. 4-OHE2 is oxidized to E2-3, 4-quinone, which can react with DNA to yield the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua, which are genotoxic and contribute to estrogen-mediated carcinogenesis [5]. Moreover, the roles of the so-called “good estrogens”, 2-OHE2 and 2-OHE1, are associated with cancer development [6]. When it comes to the role of 16α-OHE1 in cancer, there is no universal agreement. The 16α-hydroxylation pathway has been highly correlated with the growth of tumors [7]; on the other hand, 16α-OHE1 was not linked to a higher risk of cancer in a study of breast cancer patients [8]. However, 2-methoxyestradiol has shown antiproliferative and anti-angiogenic activities, and upregulates the extrinsic and intrinsic apoptotic pathways in preclinical and clinical studies [9–11].
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Authors
Endocrine Care 159
Patients and Methods
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Reagents and materials The 8 estrogens and estrogen metabolite (EM) standards are ▶ Fig. 1. Analytical reference substances, E2, E1, shown in ● 4-OHE2, 2-OHE2, 16α-OHE1, 2-MeOE2, and 2-MeOE1 were purchased from Sigma-Aldrich (Beijing, China) and 2-OHE1 from Canada-TRC (Shanghai, China). Ethynylestradiol (IS) was
obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and its purity was greater than 98 %. A polyvinylidene difluoride (PVDF) hollow fiber membrane (FoShan, China) was used in HF-LPME. The wall thickness of the fiber was 200 μm, the inner diameter was 1 000 μm, and the molecular mass cutoff was 10 000 Da.
Urine sample collection A total of 23 preoperative postmenopausal female endometrial cancer patients (confirmed by pathologic examination after surgery) were included in the study. The average age of the patients was 45–62 years. Controls were women without cancer, who had a complete healthy examination including ultrasonic examination of the liver, kidney, abdomen, and uterus at the Fourth Hospital of Hebei Medical University and tumor markers in the blood were negative. The average age of the control subjects was 46–67 years. To avoid the confounding effects of smoking, patients with a history of cigarette smoking were excluded. Twenty-four hours of urine samples were collected in 1 liter bottles containing 1 g of ascorbic acid. The collected urine samples were stored at − 20 °C until analysis. All studies were conducted according to protocols approved by the Ethics Committee at the Fourth Hospital of Hebei Medical University in China. All patients and healthy controls gave their informed consent.
Instruments, chromatographic conditions, and mass spectrometry conditions The samples were measured with a fully validated, highly selective, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method [18]. Briefly, separation was achieved with the Waters ACQUITY UPLCTM system (Waters, UK) consisting of an ACQUITY UPLC binary solvent manager and an ACQUITY UPLCTM sample manager. Analytes were analyzed with an AQUITY UPLC BEH C18 (1.7 μm; 2.1 mm × 50 mm) column, with a mobile phase consisting of mobile phase A (99.9 % acetonitrile, 0.1 % formic acid) and mobile phase B (99.9 % H2O, 0.1 % formic acid) at a flow rate of 0.5 ml/min and a temperature of 30 °C. The gradient program was as follows: 0 min, 160 % A; 6 min, 92 % A; 7–8 min, 60 % A. An injection volume of 20 μl was used. Fig. 1 Chemical structures of endogenous estrogens and their metabolites. E2: 17β-Estradiol; 4-OHE2: 4-Hydroxyestradiol; 2-OHE2: 2-Hydroxyestradiol; 2-MeOE2: 2-Methoxyestradiol; E1: Estrone; 16α-OHE1: 16α-Hydroxyestrone; 2-OHE1: 2-Hydroxyestrone; 2-MeOE1: 2-Methoxyestrone.
Zhao H et al. Endogenous Estrogen Metabolites … Horm Metab Res 2015; 47: 158–164
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The effect of estrogen metabolites on endometrial carcinogenesis remains unclear. Therefore, it is important to profile estrogens and their metabolites appropriately to provide valuable information that will aid in elucidating the mechanisms of hormone-regulated carcinogenesis and further applications in the early diagnosis, screening, prevention, and treatment of endometrial cancer. Current methods for their endogenous measurement often involve enzyme immunoassays (EIA) [12], radioimmunoassays (RIA) [13], high-performance liquid chromatography (HPLC) [14], liquid chromatography coupled with mass spectrometry detectors (LC-MS) [15], and gas chromatography mass spectrometry (GC-MS) [16]. Unfortunately, these methods are either time-consuming, irreproducible, or lack sensitivity [12–16]. The only procedure (to the best of our knowledge) is high-performance liquid chromatography (HPLC) with tandem mass spectrometry, which has shown the ability to accurately and sensitively measure estrogen metabolites. Using this method, 15 estrogen metabolites can be completely resolved and quantitatively measured in less than 10 min [17]. However, this method employs packed supercritical fluid chromatography columns for the separation of estrogen metabolites. Such a procedure would not be the best method for studying estrogens in a typical laboratory. Therefore, we explored the use of ultra-high performance liquid chromatography-mass spectrometry (UPLCMS) for the separation of estrogen metabolites. The effects of estrogens and their steroid metabolites were assessed for their association with endometrial carcinogenesis. The method used in this study showed a high sensitivity sufficient for defining the concentrations of estrogens and estrogen metabolites in urine samples from women.
160 Endocrine Care
test when the variance was uneven. A p-value < 0.05 was considered significant.
Results
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Matrix effect, mobile phase, and selectivity Electrolyte modification of the mobile phase significantly improves the electrospray interface (ESI) efficiency, resulting in enhanced response of LC-MS/MS. We used an electrolyte-free mobile phase, water-methanol (8:92). In order to identify the optimal mobile phase producing the best sensitivity, efficiency and peak shape, we tested different buffers, the result shows that the highest signal intensity was achieved when the concentration of acetic acid in the mobile phase reached 0.1 %. A postextraction spike method was carried out to investigate the matrix effect. Data showed that no suppression occurred in the ▶ Fig. 2 shows the chromatogram of the sample. In our result, ● chromatogram of urine samples from healthy females and endometrial cancer patients, respectively. The peak of E1, E2, their metabolites, and ethynylestradiol in urine samples were unambiguously identified by comparing their retention time.
Hydrolysis, extraction, and derivatization procedure A total of 140 ml urine was added to 5.6 g NaOH and 70 μl ethynylestradiol (as an internal standard), boiled for 10 min, and then centrifuged at 4 000 rpm for 10 min. The precipitate was discarded and the final volume was adjusted to 140 ml. The hollow fiber was cut into 11 cm segments. The extraction and enrichment was performed by agitation with a YL-1000 agitator (Shanghai Hengxing Company, Shanghai, China) at 500 rpm at 25 °C for 1 h. At the end of the extraction, the acceptor phase and membrane phase were transferred into a clean and dry polytef insert tube, and the elution solution evaporated to dryness at 90 °C under nitrogen gas. The residue was derivatized by the addition of 100 μl of sodium bicarbonate buffer and 100 μl of dansyl chloride solution (1 mg/ml in acetone). The sample was heated at 60 °C for 5 min to form dansyl chloride derivatives.
Preparation of standard solution Stock solutions (14.2 μg/ml) of E1, E2, 2-OHE1, 16α-OHE1, 2-OHE2, 4-OHE2, 2-MeOE1, and 2-MeOE2 were diluted to 1.42 μg/ml using HPLC grade methanol. All the standard solutions were stored at 4 °C. The stock solution was then diluted with artificial human urine to prepare a final standard solution containing 1.01 ng/ml of E1, E2, and their metabolites. A series of working solutions for E1, E2, and their metabolites were freshly prepared by diluting standard solutions with water to yield various concentrations (1.01 × 103, 808, 404, 202, 101, 50.5, 10.1 pg/ ml). A quantity of E1, E2, and their metabolites was dissolved in HPLC grade methanol to produce the IS solution at a concentration of 400 pg/ml. To analyze the standard solution, we performed measurements for the matrix effect, linearity, limits of detection (LOD) and the limits of quantification (LOQ), recovery, intra- and inter-day precisions, and stability.
Statistical analysis Statistical analysis was performed using the SPSS 13.0 software package. The normality of the data was analyzed by the test of normality. The results of the experiment were expressed as a median when the distribution of variances was abnormal. A comparison of 2 groups was performed by the nonparametric
Linearity, LOD and LOQ, and recovery The linear range and correlation coefficient for each analyte ▶ Table 1) was obtained using a weighted linear regression (● method. The limits of quantification (LOQ) and the limits of detection (LOD), defined as signal-to-noise ratios (S/N) of 10 and 3, were separately determined by 5-fold replicate analysis. The LOD was 0.05 × 10 − 4 pg/ml and the LOQ was 0.2 × 10 − 3 pg/ml. To 3 sets of 140 ml human urine, 100 μl of working standard solution for E1, E2, and their metabolites (50.5 pg/ml, 202 pg/ml, 808 pg/ml) was added, followed by the hydrolysis and extraction procedures described above. All sets of samples were derivatized ▶ Table 2 shows the recovery and and analyzed by UPLC-MS. ● relative standard deviations (RSD) of the method.
Intra- and inter-day precisions, stability To assess the repeatability and reproducibility of the newly developed method, we performed measurements for intra- and inter-day precisions with multiple concentrations. The relative standard deviations (RSD) of intra-day precision of the 3 concentrations were 10.3 %, 7.4 %, and 5.8 %. The relative standard deviations (RSD) of inter-day precision of the 3 concentrations were 11.8 %, 9.2 %, and 6.4 %. To evaluate freeze-thaw stability, samples were subjected to 3 cycles of 24-h freezing at − 20 °C and thawing at room temperature. The stability at freezing was assessed by storing samples at − 20 °C for 48 h, whereas the stability at room temperature was assessed by placing samples at room temperature for 6 h. All RSD values for the sample stability were below 9.6 %.
Analysis of human urine samples The 24-h content of urinary hormones was obtained by multiplying concentration and 24-h urine volume. The content of E2 was 198.35 μg in cases and 29.97 μg in controls. The difference was significant, as confirmed by the nonparametric test (p < 0.001). The content of 4-OHE2 was high and the content of 2-MeOE1 was low, but the distribution of 24-h hormone content ▶ Fig. 3 and was abnormal. The data are shown as medians (● Table 1S). Overall, the levels of E2, 4-OHE2, 2-MeOE2, and 2-MeOE1 were significantly different between the 2 groups. The
Zhao H et al. Endogenous Estrogen Metabolites … Horm Metab Res 2015; 47: 158–164
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A triple quadrupole mass spectrometer (TQ-S, Waters, UK), equipped with an electrospray ionization source, was used for the quantification of target analytes and the analyses were performed in positive mode. The mass spectrometer was operated in multiple reactions monitoring (MRM) mode, which was employed for quantification. Masslynx software (Waters, UK) was used to collect and analyze all data. MRM product ions were optimized using positive electrospray interface (ESI). We found that the positive electrospray ionization could offer better sensitivity and reproducibility. In the full scan mass spectra, the protonated molecular ions [M + H] + of E1, E2, 2-OHE1, 16α-OHE1, 2-OHE2, 4-OHE2, 2-MeOE1, 2-MeOE2, and IS (m/z = 504.2, 506.1, 753.1, 520.0, 755.1, 755.1, 534.2, 536.0, 530.3) were stable and highly abundant. Under the product ion scan mode, the most intensive product ions were m/z 171.1 from 504.2, m/z 171.0 from 506.1, m/z 170.1 from 753.1, m/z 171.2 from 520.0, m/z 170.0 from 755.1, m/z 170.0 from 755.1, m/z 171.0 from 534.2, m/z 171.0 from 536.0, and m/z 171.3 from 530.3. In order to obtain a higher signal for both precursor ions and product ions, we optimized the mass spectrometric parameters.
Endocrine Care 161
b 100
100
2.2
E2
2.2
0 100
%
E2
%
0 100 3.3
3.27 0
0
100
100
0
0
100
100 2-MeOE2
%
2-MeOE2
0
0 100
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2.5
E1
%
%
E1
2.2 0
0 100 2-OHE1
3.6
%
%
100
3.6
100
2.4
%
2-MeOE1
2-MeOE1
0
0 100
100
16α-OHE1 %
2.4
%
100
16α-OHE1
%
1.3 0
1.3 0
2.4
100
2.4
%
Ethynylestradiol
%
2-OHE1
0
0
100
2-OHE2
2.1
2.1 %
3.4
%
%
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2-OHE2
4-OHE2
Ethynylestradiol 0
0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Time (mins)
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 Time (mins)
Fig. 2 The chromatogram of urine samples from healthy women a and endometrial cancer patients b after extraction with LC-MS. The Arabic numbers mean the retention time of estrogens and their metabolites. E2: 17β-Estradiol; 4-OHE2: 4-Hydroxyestradiol; 2-OHE2: 2-Hydroxyestradiol; 2-MeOE2: 2-Methoxyestradiol; E1: Estrone; 16α-OHE1: 16α-Hydroxyestrone; 2-OHE1: 2-Hydroxyestrone; 2-MeOE1: 2-Methoxyestrone, and IS: Ethynylestradiol.
endometrial cancer patients excreted greater amounts of E2 and 4-OHE2 (p < 0.001, p < 0.001), but lesser amounts of 2-MeOE2 and 2-MeOE1 compared with healthy females (p < 0.001, p < 0.01). Other hormones were found at levels deemed insignificant, including E1, with a high tendency (p > 0.05).
significantly different between the 2 groups. The levels of 2-OHE2/2-MeOE2, 2-OHE1/2-MeOE1, E1/2-OHE1, and E1/16αOHE1, were not different.
Discussion Relationships between estrogens and their metabolites
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E1 and E2 were converted into hydroxyestrogens and methoxyestrogens through the 2-, 4-, and 16α-pathways. The specific ▶ Fig. 4. ratios between 8 hormones in the urine are displayed in ● The levels of E2/E1, E2/2-MeOE2, and 4-OHE2/2-MeOE2 were
Estrogen is a known risk factor and plays an important role in the etiology of endometrial cancer. Two estrogens and their 6 metabolites were measured with HF-LPME by high-performance liquid chromatography-mass spectrometry in urine samples
Zhao H et al. Endogenous Estrogen Metabolites … Horm Metab Res 2015; 47: 158–164
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%
4-OHE2
%
a
from endometrial cancer patients and healthy women. In this study, the 24-h content of 17β-estradiol (E2) was high in the cases, which was associated with endometrial carcinogenesis. It has been reported that E2, via activation of the PI3K/AKT and MPAK signal pathways, contributes to enhance endometrial cancer cell proliferation and invasion [19]. The first step of estradiol metabolism is its conversion to estrone by oxidation at the C17 position, a reversible process. Estrone (E1) and estradiol (E2) can be transformed into one another in the human body through 17β-hydroxysteroid dehydrogenases (17β-HSD), which include 17β-hydroxysteroid dehydrogenase Table 1 Summary of linear regression for calibration curves and the limits of detection (LOD) and the limits of quantification (LOQ). Analyte
Slope
Intercept
R2
LOD (pg/ml)
LOQ (pg/ml)
E2 4-OHE2 2-OHE2 2-MeOE2 E1 16α-OHE1 2-OHE1 2-MeOE1
0.0592 0.0218 0.0535 0.0315 0.0464 0.1657 0.0389 0.0116
0.0024 0.0046 0.0089 0.0077 0.0068 0.0019 0.0062 0.0039
0.9925 0.9942 0.9960 0.9914 0.9973 0.9936 0.9988 0.9953
0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4
0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3
E2: 17β-Estradiol; 4-OHE2: 4-Hydroxyestradiol; 2-OHE2: 2-Hydroxyestradiol; 2-MeOE2: 2-Methoxyestradiol; E1: Estrone; 16α-OHE1: 16α-Hydroxyestrone; 2-OHE1: 2-Hydroxyestrone; 2-MeOE1: 2-Methoxyestrone
Table 2 The results of the recovery ( %) and relative standard deviations (RSD %). Analyte
50.5 pg/ml
E2 4-OHE2 2-OHE2 2-MeOE2 E1 16α-OHE1 2-OHE1 2-MeOE1
202 pg/ml
808 pg/ml
Recovery
RSD
Recovery
RSD
Recovery
RSD
94 94 95 91 92 91 89 90
9.5 9.2 10.5 9.4 9.9 8.2 10.2 8.8
103 96 92 95 94 93 94 93
6.8 7.9 8.3 7.5 8.4 6.7 7.3 6.8
97 105 97 105 104 106 95 94
6.4 5.1 7.3 6.1 5.5 5.8 5.3 6.3
For abbreviations, see ● ▶ Table 1
type 1 (17β-HSD-1) and type 2 (17β-HSD-2). The transformation of E2 to E1 by 17β-HSD-2 is dominant in normal tissue, while 17β-HSD-1 transformation of E1 to active E2 is predominant in endometrial cancer tissue [20]. E1-induced activation of estrogen signaling is similar to E2, but E1 has low activity. In the study, the value of E2/E1 in the case group was higher than the normal group (p < 0.01). Our results show that the transformation of E1 to E2 is a major step in endometrial carcinoma patients. 4-OHE2 plays an important role in estrogen-mediated carcinogenesis. 4-OHE2 increases the risk of endometrial cancer in rats more than 9 times compared with E2 [21]. In this study, the amount of 4-OHE2 in the urine of patients with endometrial cancer was also significantly higher than the control group, indicating the close relationship of 4-OHE2 with endometrial cancer. The high level of 4-OHE2 was due to cytochrome P450 1B1 (CYP1B1) to some extent [22]. It has been reported that the expression of CYP1B1 is upregulated in endometrial cancers [23]. 2-Methoxyestradiol (2-MeOE2), a naturally occurring metabolite of estradiol, is highly cytotoxic to a wide range of tumor cells but harmless to most normal cells. 2-Methoxyestrone (2-MeOE1), the estrone analogue of 2-MeOE2, induces cell death and increases the gene expression of cytokines implicated in inhibiting differentiation and inducing apoptosis [24]. In our study, the 24-h content of 2-MeOE2 (E1) was low in the urine samples of endometrial cancer patients (p < 0.01, p < 0.01), which was associated with a reduction in their anticancer activity. Changes in catechol-O-methyltransferase (COMT) may explain the above process, as reduced COMT activity has been suggested as a risk factor for estrogen-associated cancers [21, 25]. In this study, the concentration of 16α-hydroxyestrone (16αOHE1), 2-hydroxyestradiol (2-OHE2), and 2-hydroxyestrone (2-OHE1) were also measured in human urine samples. Their effects on carcinogenesis are controversial [6, 8]. In the present study, the 24-h content of 16a-OHE1, 2-OHE2, and 2-OHE1 in the urine samples of patients with endometrial carcinoma were nearly equal to the healthy women. Thus, the levels of 16αOHE1, 2-OHE2, and 2-OHE1 were not linked to a higher risk of cancer in the study of endometrial cancer patients. The effects of 16α-OHE1, 2-OHE2, and 2-OHE1 on endometrial carcinogenesis still need further investigation.
Fig. 3 The 24-h content of urinary estrogens a and their metabolites b excretion in healthy women (Controls n = 23) and endometrial cancer patients (Cases n = 23). The distribution of date was unbalanced, so the date was shown as the median. The statistics was performed by nonparametric test. *p < 0.01. E2: 17β-Estradiol; 4-OHE2: 4-Hydroxyestradiol; 2-OHE2: 2-Hydroxyestradiol; 2-MeOE2: 2-Methoxyestradiol; E1: Estrone; 16α-OHE1: 16α-Hydroxyestrone; 2-OHE1: 2-Hydroxyestrone; 2-MeOE1: 2-Methoxyestrone.
a Urinary estrogen content of 24-h (ug)
300
Controls
250
*
200 150 100 50 0
E2
b 25
Urinary estrogen content of 24-h (ng)
Cases
20
E1 Controls
Cases
*
15 10
*
5 0
4-OHE2
2-OHE2
2-MeOE2
* 16α-OHE1
2-OHE1
Zhao H et al. Endogenous Estrogen Metabolites … Horm Metab Res 2015; 47: 158–164
2-MeOE1
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162 Endocrine Care
40 35 30 25 20 15 10 5 0
Controls
*
*
* E2/E1
Cases
E2/ 4-OHE2/ 2-OHE2/ E1/ E1/ 2-MeOE2 2-MeOE2 2-MeOE2 16a-OHE1 2-MeOE1
To explore the details of estrogen metabolism, the ratio of estrogens and their metabolites was compared between 2 groups (Table 2S). On the basis of our results, the level of E2/E1, E2/2MeOE2, and 4-OHE2/2-MeOE2 was significantly higher in the ▶ Fig. 4), indiendometrial cancer patients than in the controls (● cating that the excretory pattern of estrogen metabolites and the carcinogenic effect is stronger than the protective effect, ultimately promoting cancer. Thus, estrogens and their normal metabolism are quite important for the proliferation and differentiation of endometrial cells. 4-OHE2 and 2-MeOE2 are key reagents in the process of endometrial carcinogenesis, during which key enzymes, such as CYP1B1 and COMT, play an important role. The ratio of E2/E1, E2/2-MeOE2, and 4-OHE2/2-MeOE2 may thus potentially serve as indicators of endometrial carcinogenesis. In our present study, we have developed an LC-MS based technique to measure estrogens and their metabolites in urine samples. Although the method is convenient and cheap, much more human urine and a longer test time were needed. In view of the chemical structures and the metabolic pathways involved, high levels of estradiol (E2) and 4-hydroxyestradiol (4-OHE2) as well as a low level of 2-methoxyestrone (2-MeOE1) and 2-methoxyestradiol (2-MeOE2) in endometrial cancer cases implied an imbalance among the hydroxylation and methylation pathways. Estradiol (E2) and 4-hydroxyestradiol (4-OHE2) might enhance the promotion of endometrial cancer, which coincided with the currently known quinone-mediated carcinogenesis mechanism. The lower excretion of 2-MeOE1 and 2-MeOE2 in the urine of endometrial cancer patients indicated that the methylation pathway may play an important role in the inhibition of endometrial cancer. The results from this study could help elucidate the association of the estrogen metabolic pathway with carcinogenesis and could also provide potential biomarkers for the assessment of estrogen-induced breast cancer risk. The newly developed method could help to practically aid the clinician in the diagnostic workup for endometrial cancer.
Acknowledgements
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This work was supported by the Hebei medical science research (number: 20100118) and by funds for the Construction of Potentially Distinguished Scientific Projects in the program of Hebei Universities. We would like to thank the Department of Gynecology of the 4th hospital and the Institute of Hebei Cancer Research. We are especially grateful to Ye Jiang and Yan Liu for their excellent technical assistance. We are also grateful to the patients and healthy controls for agreeing to participate in this study.
16aOHE1/ 2-OHE1
2-OHE1/ 2-MeOE1
Fig. 4 Relationship between parent estrogen and its metabolite. The specific value between estrogens and their metabolites in the urine were displayed in healthy women (Controls n = 23) and endometrial cancer patients (Cases n = 23). The distribution of date was unbalanced; the date was shown as the median. The statistics was performed by nonparametric test. *p < 0.05. E2: 17β-Estradiol; 4-OHE2: 4-Hydroxyestradiol; 2-OHE2: 2-Hydroxyestradiol; 2-MeOE2: 2-Methoxyestradiol; E1: Estrone; 16α-OHE1: 16α-Hydroxyestrone; 2-OHE1: 2-Hydroxyestrone; 2-MeOE1: 2-Methoxyestrone.
Conflict of Interest
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The authors declare that they have no conflicts of interest in the authorship or publication of this contribution.
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Endogenous Estrogen Metabolites as Biomarkers for Endometrial Cancer via a Novel Method of Liquid Chromatography-Mass Spectrometry with Hollow Fiber Liquid-Phase Microextraction
Affiliations
Key words ▶ estrogens ● ▶ 4-hydroxyestradiol ● ▶ 2-methoxyestradiol ● ▶ endometrial cancer ● ▶ LC-MS ●
received 19.06.2013 accepted 06.03.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1371865 Published online: April 10, 2014 Horm Metab Res 2015; 47: 158–164 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence L. Li, MD, PhD Department of Gynecology The Fourth Hospital of Hebei Medical University Hebei Province 050000 P. R. China Tel.: + 86/311/86095 627 Fax: + 86/311/86095 588 [email protected]
H. Zhao1, Y. Jiang2, Y. Liu2, C. Yun1, L. Li1 1 2
Department of Gynecology, The Fourth Hospital of Hebei Medical University, Hebei Province, P. R. China Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Hebei Province, P. R. China
Abstract
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Increased levels of endogenous estrogens and their metabolites are well-known risk factors of endometrial cancer. The aim of this study was to quantitatively assess the potential for estrogen metabolites to serve as biomarkers of endometrial carcinogenesis. The following estrogen metabolites were evaluated: 2-hydroxyestradiol (2-OHE2), 2-hydroxyestrone (2-OHE1), 4-hydroxyestradiol (4-OHE2), 4-hydroxyestrone (4-OHE1), 16α-hydroxyestrone (16α-OHE1), 2-methoxyestradiol (2-MeOE2), and 2-methoxyestrone (2-MeOE1). The low content of estrogen metabolites in urine makes their measurement difficult. To address this issue, we developed a rapid, sensitive, specific, and accurate liquid chromatography-mass spectrometry (LC-MS) method, with hollow fiber liquid-phase micro-extraction (HF-
Introduction
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Endometrial carcinoma is the most common malignancy of the female genital tract and is increasing in frequency [1]. The risk of endometrial carcinoma is related to a lifelong exposure to estrogen. Estrogen plays an important role in the genesis of human endometrial cancer by increasing the mitotic activity of endometrial cells. 17β-Estradiol (E2) and estrone (E1), parent estrogens in the estrogen metabolic pathways, mainly induce tumors by stimulation of epithelial cell proliferation or the formation of genotoxic catechol estrogen-quinones [2]. Catechol estrogens, natural active metabolites of estrogens, are produced by hydroxylation of estradiol and estrone at the C-2, C-4, and C-16a positions. Catechol estrogens include 2-hydroxyestradiol (2-OHE2), 2-hydroxyestrone (2-OHE1), 4-hydroxyestradiol (4-OHE2), 4-hydroxyestrone (4-OHE1), and 16α-hydroxyestrone (16α-OHE1) [2, 3]. 2-OHE2 and 2-OHE1 are methylated to 2-MeOE2 and
Zhao H et al. Endogenous Estrogen Metabolites … Horm Metab Res 2015; 47: 158–164
LPME) for an enriched pretreatment of the sample and for the simultaneous quantification of estrogens and their metabolites in the urine samples of 23 post-menopausal female endometrial cancer patients and 23 post-menopausal healthy female controls. The levels of estrogens were found to differ between the endometrial cancer patients and the controls. The level of 4-OHE2 was elevated in patients compared with the controls, while the levels of 2-MeOE1 and 2-MeOE2 were reduced in the endometrial cancer group. The results of this study indicate an imbalance of estrogen metabolites in endometrial carcinogenesis, and that the elevation of 4-OHE2 may be used as a potential biomarker for the risk assessment of estrogen-induced endometrial cancer. Supporting Information for this article is available online at http://www.thieme-connect.de/ products.
2-MeOE1 by the enzyme catechol-O-methyltransferase (COMT) [4]. Meanwhile, 4-OHE2 plays an important role in estrogen-mediated carcinogenesis. 4-OHE2 is oxidized to E2-3, 4-quinone, which can react with DNA to yield the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua, which are genotoxic and contribute to estrogen-mediated carcinogenesis [5]. Moreover, the roles of the so-called “good estrogens”, 2-OHE2 and 2-OHE1, are associated with cancer development [6]. When it comes to the role of 16α-OHE1 in cancer, there is no universal agreement. The 16α-hydroxylation pathway has been highly correlated with the growth of tumors [7]; on the other hand, 16α-OHE1 was not linked to a higher risk of cancer in a study of breast cancer patients [8]. However, 2-methoxyestradiol has shown antiproliferative and anti-angiogenic activities, and upregulates the extrinsic and intrinsic apoptotic pathways in preclinical and clinical studies [9–11].
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Authors
Endocrine Care 159
Patients and Methods
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Reagents and materials The 8 estrogens and estrogen metabolite (EM) standards are ▶ Fig. 1. Analytical reference substances, E2, E1, shown in ● 4-OHE2, 2-OHE2, 16α-OHE1, 2-MeOE2, and 2-MeOE1 were purchased from Sigma-Aldrich (Beijing, China) and 2-OHE1 from Canada-TRC (Shanghai, China). Ethynylestradiol (IS) was
obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and its purity was greater than 98 %. A polyvinylidene difluoride (PVDF) hollow fiber membrane (FoShan, China) was used in HF-LPME. The wall thickness of the fiber was 200 μm, the inner diameter was 1 000 μm, and the molecular mass cutoff was 10 000 Da.
Urine sample collection A total of 23 preoperative postmenopausal female endometrial cancer patients (confirmed by pathologic examination after surgery) were included in the study. The average age of the patients was 45–62 years. Controls were women without cancer, who had a complete healthy examination including ultrasonic examination of the liver, kidney, abdomen, and uterus at the Fourth Hospital of Hebei Medical University and tumor markers in the blood were negative. The average age of the control subjects was 46–67 years. To avoid the confounding effects of smoking, patients with a history of cigarette smoking were excluded. Twenty-four hours of urine samples were collected in 1 liter bottles containing 1 g of ascorbic acid. The collected urine samples were stored at − 20 °C until analysis. All studies were conducted according to protocols approved by the Ethics Committee at the Fourth Hospital of Hebei Medical University in China. All patients and healthy controls gave their informed consent.
Instruments, chromatographic conditions, and mass spectrometry conditions The samples were measured with a fully validated, highly selective, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method [18]. Briefly, separation was achieved with the Waters ACQUITY UPLCTM system (Waters, UK) consisting of an ACQUITY UPLC binary solvent manager and an ACQUITY UPLCTM sample manager. Analytes were analyzed with an AQUITY UPLC BEH C18 (1.7 μm; 2.1 mm × 50 mm) column, with a mobile phase consisting of mobile phase A (99.9 % acetonitrile, 0.1 % formic acid) and mobile phase B (99.9 % H2O, 0.1 % formic acid) at a flow rate of 0.5 ml/min and a temperature of 30 °C. The gradient program was as follows: 0 min, 160 % A; 6 min, 92 % A; 7–8 min, 60 % A. An injection volume of 20 μl was used. Fig. 1 Chemical structures of endogenous estrogens and their metabolites. E2: 17β-Estradiol; 4-OHE2: 4-Hydroxyestradiol; 2-OHE2: 2-Hydroxyestradiol; 2-MeOE2: 2-Methoxyestradiol; E1: Estrone; 16α-OHE1: 16α-Hydroxyestrone; 2-OHE1: 2-Hydroxyestrone; 2-MeOE1: 2-Methoxyestrone.
Zhao H et al. Endogenous Estrogen Metabolites … Horm Metab Res 2015; 47: 158–164
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The effect of estrogen metabolites on endometrial carcinogenesis remains unclear. Therefore, it is important to profile estrogens and their metabolites appropriately to provide valuable information that will aid in elucidating the mechanisms of hormone-regulated carcinogenesis and further applications in the early diagnosis, screening, prevention, and treatment of endometrial cancer. Current methods for their endogenous measurement often involve enzyme immunoassays (EIA) [12], radioimmunoassays (RIA) [13], high-performance liquid chromatography (HPLC) [14], liquid chromatography coupled with mass spectrometry detectors (LC-MS) [15], and gas chromatography mass spectrometry (GC-MS) [16]. Unfortunately, these methods are either time-consuming, irreproducible, or lack sensitivity [12–16]. The only procedure (to the best of our knowledge) is high-performance liquid chromatography (HPLC) with tandem mass spectrometry, which has shown the ability to accurately and sensitively measure estrogen metabolites. Using this method, 15 estrogen metabolites can be completely resolved and quantitatively measured in less than 10 min [17]. However, this method employs packed supercritical fluid chromatography columns for the separation of estrogen metabolites. Such a procedure would not be the best method for studying estrogens in a typical laboratory. Therefore, we explored the use of ultra-high performance liquid chromatography-mass spectrometry (UPLCMS) for the separation of estrogen metabolites. The effects of estrogens and their steroid metabolites were assessed for their association with endometrial carcinogenesis. The method used in this study showed a high sensitivity sufficient for defining the concentrations of estrogens and estrogen metabolites in urine samples from women.
160 Endocrine Care
test when the variance was uneven. A p-value < 0.05 was considered significant.
Results
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Matrix effect, mobile phase, and selectivity Electrolyte modification of the mobile phase significantly improves the electrospray interface (ESI) efficiency, resulting in enhanced response of LC-MS/MS. We used an electrolyte-free mobile phase, water-methanol (8:92). In order to identify the optimal mobile phase producing the best sensitivity, efficiency and peak shape, we tested different buffers, the result shows that the highest signal intensity was achieved when the concentration of acetic acid in the mobile phase reached 0.1 %. A postextraction spike method was carried out to investigate the matrix effect. Data showed that no suppression occurred in the ▶ Fig. 2 shows the chromatogram of the sample. In our result, ● chromatogram of urine samples from healthy females and endometrial cancer patients, respectively. The peak of E1, E2, their metabolites, and ethynylestradiol in urine samples were unambiguously identified by comparing their retention time.
Hydrolysis, extraction, and derivatization procedure A total of 140 ml urine was added to 5.6 g NaOH and 70 μl ethynylestradiol (as an internal standard), boiled for 10 min, and then centrifuged at 4 000 rpm for 10 min. The precipitate was discarded and the final volume was adjusted to 140 ml. The hollow fiber was cut into 11 cm segments. The extraction and enrichment was performed by agitation with a YL-1000 agitator (Shanghai Hengxing Company, Shanghai, China) at 500 rpm at 25 °C for 1 h. At the end of the extraction, the acceptor phase and membrane phase were transferred into a clean and dry polytef insert tube, and the elution solution evaporated to dryness at 90 °C under nitrogen gas. The residue was derivatized by the addition of 100 μl of sodium bicarbonate buffer and 100 μl of dansyl chloride solution (1 mg/ml in acetone). The sample was heated at 60 °C for 5 min to form dansyl chloride derivatives.
Preparation of standard solution Stock solutions (14.2 μg/ml) of E1, E2, 2-OHE1, 16α-OHE1, 2-OHE2, 4-OHE2, 2-MeOE1, and 2-MeOE2 were diluted to 1.42 μg/ml using HPLC grade methanol. All the standard solutions were stored at 4 °C. The stock solution was then diluted with artificial human urine to prepare a final standard solution containing 1.01 ng/ml of E1, E2, and their metabolites. A series of working solutions for E1, E2, and their metabolites were freshly prepared by diluting standard solutions with water to yield various concentrations (1.01 × 103, 808, 404, 202, 101, 50.5, 10.1 pg/ ml). A quantity of E1, E2, and their metabolites was dissolved in HPLC grade methanol to produce the IS solution at a concentration of 400 pg/ml. To analyze the standard solution, we performed measurements for the matrix effect, linearity, limits of detection (LOD) and the limits of quantification (LOQ), recovery, intra- and inter-day precisions, and stability.
Statistical analysis Statistical analysis was performed using the SPSS 13.0 software package. The normality of the data was analyzed by the test of normality. The results of the experiment were expressed as a median when the distribution of variances was abnormal. A comparison of 2 groups was performed by the nonparametric
Linearity, LOD and LOQ, and recovery The linear range and correlation coefficient for each analyte ▶ Table 1) was obtained using a weighted linear regression (● method. The limits of quantification (LOQ) and the limits of detection (LOD), defined as signal-to-noise ratios (S/N) of 10 and 3, were separately determined by 5-fold replicate analysis. The LOD was 0.05 × 10 − 4 pg/ml and the LOQ was 0.2 × 10 − 3 pg/ml. To 3 sets of 140 ml human urine, 100 μl of working standard solution for E1, E2, and their metabolites (50.5 pg/ml, 202 pg/ml, 808 pg/ml) was added, followed by the hydrolysis and extraction procedures described above. All sets of samples were derivatized ▶ Table 2 shows the recovery and and analyzed by UPLC-MS. ● relative standard deviations (RSD) of the method.
Intra- and inter-day precisions, stability To assess the repeatability and reproducibility of the newly developed method, we performed measurements for intra- and inter-day precisions with multiple concentrations. The relative standard deviations (RSD) of intra-day precision of the 3 concentrations were 10.3 %, 7.4 %, and 5.8 %. The relative standard deviations (RSD) of inter-day precision of the 3 concentrations were 11.8 %, 9.2 %, and 6.4 %. To evaluate freeze-thaw stability, samples were subjected to 3 cycles of 24-h freezing at − 20 °C and thawing at room temperature. The stability at freezing was assessed by storing samples at − 20 °C for 48 h, whereas the stability at room temperature was assessed by placing samples at room temperature for 6 h. All RSD values for the sample stability were below 9.6 %.
Analysis of human urine samples The 24-h content of urinary hormones was obtained by multiplying concentration and 24-h urine volume. The content of E2 was 198.35 μg in cases and 29.97 μg in controls. The difference was significant, as confirmed by the nonparametric test (p < 0.001). The content of 4-OHE2 was high and the content of 2-MeOE1 was low, but the distribution of 24-h hormone content ▶ Fig. 3 and was abnormal. The data are shown as medians (● Table 1S). Overall, the levels of E2, 4-OHE2, 2-MeOE2, and 2-MeOE1 were significantly different between the 2 groups. The
Zhao H et al. Endogenous Estrogen Metabolites … Horm Metab Res 2015; 47: 158–164
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A triple quadrupole mass spectrometer (TQ-S, Waters, UK), equipped with an electrospray ionization source, was used for the quantification of target analytes and the analyses were performed in positive mode. The mass spectrometer was operated in multiple reactions monitoring (MRM) mode, which was employed for quantification. Masslynx software (Waters, UK) was used to collect and analyze all data. MRM product ions were optimized using positive electrospray interface (ESI). We found that the positive electrospray ionization could offer better sensitivity and reproducibility. In the full scan mass spectra, the protonated molecular ions [M + H] + of E1, E2, 2-OHE1, 16α-OHE1, 2-OHE2, 4-OHE2, 2-MeOE1, 2-MeOE2, and IS (m/z = 504.2, 506.1, 753.1, 520.0, 755.1, 755.1, 534.2, 536.0, 530.3) were stable and highly abundant. Under the product ion scan mode, the most intensive product ions were m/z 171.1 from 504.2, m/z 171.0 from 506.1, m/z 170.1 from 753.1, m/z 171.2 from 520.0, m/z 170.0 from 755.1, m/z 170.0 from 755.1, m/z 171.0 from 534.2, m/z 171.0 from 536.0, and m/z 171.3 from 530.3. In order to obtain a higher signal for both precursor ions and product ions, we optimized the mass spectrometric parameters.
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b 100
100
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Ethynylestradiol 0
0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Time (mins)
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 Time (mins)
Fig. 2 The chromatogram of urine samples from healthy women a and endometrial cancer patients b after extraction with LC-MS. The Arabic numbers mean the retention time of estrogens and their metabolites. E2: 17β-Estradiol; 4-OHE2: 4-Hydroxyestradiol; 2-OHE2: 2-Hydroxyestradiol; 2-MeOE2: 2-Methoxyestradiol; E1: Estrone; 16α-OHE1: 16α-Hydroxyestrone; 2-OHE1: 2-Hydroxyestrone; 2-MeOE1: 2-Methoxyestrone, and IS: Ethynylestradiol.
endometrial cancer patients excreted greater amounts of E2 and 4-OHE2 (p < 0.001, p < 0.001), but lesser amounts of 2-MeOE2 and 2-MeOE1 compared with healthy females (p < 0.001, p < 0.01). Other hormones were found at levels deemed insignificant, including E1, with a high tendency (p > 0.05).
significantly different between the 2 groups. The levels of 2-OHE2/2-MeOE2, 2-OHE1/2-MeOE1, E1/2-OHE1, and E1/16αOHE1, were not different.
Discussion Relationships between estrogens and their metabolites
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E1 and E2 were converted into hydroxyestrogens and methoxyestrogens through the 2-, 4-, and 16α-pathways. The specific ▶ Fig. 4. ratios between 8 hormones in the urine are displayed in ● The levels of E2/E1, E2/2-MeOE2, and 4-OHE2/2-MeOE2 were
Estrogen is a known risk factor and plays an important role in the etiology of endometrial cancer. Two estrogens and their 6 metabolites were measured with HF-LPME by high-performance liquid chromatography-mass spectrometry in urine samples
Zhao H et al. Endogenous Estrogen Metabolites … Horm Metab Res 2015; 47: 158–164
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%
4-OHE2
%
a
from endometrial cancer patients and healthy women. In this study, the 24-h content of 17β-estradiol (E2) was high in the cases, which was associated with endometrial carcinogenesis. It has been reported that E2, via activation of the PI3K/AKT and MPAK signal pathways, contributes to enhance endometrial cancer cell proliferation and invasion [19]. The first step of estradiol metabolism is its conversion to estrone by oxidation at the C17 position, a reversible process. Estrone (E1) and estradiol (E2) can be transformed into one another in the human body through 17β-hydroxysteroid dehydrogenases (17β-HSD), which include 17β-hydroxysteroid dehydrogenase Table 1 Summary of linear regression for calibration curves and the limits of detection (LOD) and the limits of quantification (LOQ). Analyte
Slope
Intercept
R2
LOD (pg/ml)
LOQ (pg/ml)
E2 4-OHE2 2-OHE2 2-MeOE2 E1 16α-OHE1 2-OHE1 2-MeOE1
0.0592 0.0218 0.0535 0.0315 0.0464 0.1657 0.0389 0.0116
0.0024 0.0046 0.0089 0.0077 0.0068 0.0019 0.0062 0.0039
0.9925 0.9942 0.9960 0.9914 0.9973 0.9936 0.9988 0.9953
0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4 0.05 × 10 − 4
0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3 0.2 × 10 − 3
E2: 17β-Estradiol; 4-OHE2: 4-Hydroxyestradiol; 2-OHE2: 2-Hydroxyestradiol; 2-MeOE2: 2-Methoxyestradiol; E1: Estrone; 16α-OHE1: 16α-Hydroxyestrone; 2-OHE1: 2-Hydroxyestrone; 2-MeOE1: 2-Methoxyestrone
Table 2 The results of the recovery ( %) and relative standard deviations (RSD %). Analyte
50.5 pg/ml
E2 4-OHE2 2-OHE2 2-MeOE2 E1 16α-OHE1 2-OHE1 2-MeOE1
202 pg/ml
808 pg/ml
Recovery
RSD
Recovery
RSD
Recovery
RSD
94 94 95 91 92 91 89 90
9.5 9.2 10.5 9.4 9.9 8.2 10.2 8.8
103 96 92 95 94 93 94 93
6.8 7.9 8.3 7.5 8.4 6.7 7.3 6.8
97 105 97 105 104 106 95 94
6.4 5.1 7.3 6.1 5.5 5.8 5.3 6.3
For abbreviations, see ● ▶ Table 1
type 1 (17β-HSD-1) and type 2 (17β-HSD-2). The transformation of E2 to E1 by 17β-HSD-2 is dominant in normal tissue, while 17β-HSD-1 transformation of E1 to active E2 is predominant in endometrial cancer tissue [20]. E1-induced activation of estrogen signaling is similar to E2, but E1 has low activity. In the study, the value of E2/E1 in the case group was higher than the normal group (p < 0.01). Our results show that the transformation of E1 to E2 is a major step in endometrial carcinoma patients. 4-OHE2 plays an important role in estrogen-mediated carcinogenesis. 4-OHE2 increases the risk of endometrial cancer in rats more than 9 times compared with E2 [21]. In this study, the amount of 4-OHE2 in the urine of patients with endometrial cancer was also significantly higher than the control group, indicating the close relationship of 4-OHE2 with endometrial cancer. The high level of 4-OHE2 was due to cytochrome P450 1B1 (CYP1B1) to some extent [22]. It has been reported that the expression of CYP1B1 is upregulated in endometrial cancers [23]. 2-Methoxyestradiol (2-MeOE2), a naturally occurring metabolite of estradiol, is highly cytotoxic to a wide range of tumor cells but harmless to most normal cells. 2-Methoxyestrone (2-MeOE1), the estrone analogue of 2-MeOE2, induces cell death and increases the gene expression of cytokines implicated in inhibiting differentiation and inducing apoptosis [24]. In our study, the 24-h content of 2-MeOE2 (E1) was low in the urine samples of endometrial cancer patients (p < 0.01, p < 0.01), which was associated with a reduction in their anticancer activity. Changes in catechol-O-methyltransferase (COMT) may explain the above process, as reduced COMT activity has been suggested as a risk factor for estrogen-associated cancers [21, 25]. In this study, the concentration of 16α-hydroxyestrone (16αOHE1), 2-hydroxyestradiol (2-OHE2), and 2-hydroxyestrone (2-OHE1) were also measured in human urine samples. Their effects on carcinogenesis are controversial [6, 8]. In the present study, the 24-h content of 16a-OHE1, 2-OHE2, and 2-OHE1 in the urine samples of patients with endometrial carcinoma were nearly equal to the healthy women. Thus, the levels of 16αOHE1, 2-OHE2, and 2-OHE1 were not linked to a higher risk of cancer in the study of endometrial cancer patients. The effects of 16α-OHE1, 2-OHE2, and 2-OHE1 on endometrial carcinogenesis still need further investigation.
Fig. 3 The 24-h content of urinary estrogens a and their metabolites b excretion in healthy women (Controls n = 23) and endometrial cancer patients (Cases n = 23). The distribution of date was unbalanced, so the date was shown as the median. The statistics was performed by nonparametric test. *p < 0.01. E2: 17β-Estradiol; 4-OHE2: 4-Hydroxyestradiol; 2-OHE2: 2-Hydroxyestradiol; 2-MeOE2: 2-Methoxyestradiol; E1: Estrone; 16α-OHE1: 16α-Hydroxyestrone; 2-OHE1: 2-Hydroxyestrone; 2-MeOE1: 2-Methoxyestrone.
a Urinary estrogen content of 24-h (ug)
300
Controls
250
*
200 150 100 50 0
E2
b 25
Urinary estrogen content of 24-h (ng)
Cases
20
E1 Controls
Cases
*
15 10
*
5 0
4-OHE2
2-OHE2
2-MeOE2
* 16α-OHE1
2-OHE1
Zhao H et al. Endogenous Estrogen Metabolites … Horm Metab Res 2015; 47: 158–164
2-MeOE1
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162 Endocrine Care
40 35 30 25 20 15 10 5 0
Controls
*
*
* E2/E1
Cases
E2/ 4-OHE2/ 2-OHE2/ E1/ E1/ 2-MeOE2 2-MeOE2 2-MeOE2 16a-OHE1 2-MeOE1
To explore the details of estrogen metabolism, the ratio of estrogens and their metabolites was compared between 2 groups (Table 2S). On the basis of our results, the level of E2/E1, E2/2MeOE2, and 4-OHE2/2-MeOE2 was significantly higher in the ▶ Fig. 4), indiendometrial cancer patients than in the controls (● cating that the excretory pattern of estrogen metabolites and the carcinogenic effect is stronger than the protective effect, ultimately promoting cancer. Thus, estrogens and their normal metabolism are quite important for the proliferation and differentiation of endometrial cells. 4-OHE2 and 2-MeOE2 are key reagents in the process of endometrial carcinogenesis, during which key enzymes, such as CYP1B1 and COMT, play an important role. The ratio of E2/E1, E2/2-MeOE2, and 4-OHE2/2-MeOE2 may thus potentially serve as indicators of endometrial carcinogenesis. In our present study, we have developed an LC-MS based technique to measure estrogens and their metabolites in urine samples. Although the method is convenient and cheap, much more human urine and a longer test time were needed. In view of the chemical structures and the metabolic pathways involved, high levels of estradiol (E2) and 4-hydroxyestradiol (4-OHE2) as well as a low level of 2-methoxyestrone (2-MeOE1) and 2-methoxyestradiol (2-MeOE2) in endometrial cancer cases implied an imbalance among the hydroxylation and methylation pathways. Estradiol (E2) and 4-hydroxyestradiol (4-OHE2) might enhance the promotion of endometrial cancer, which coincided with the currently known quinone-mediated carcinogenesis mechanism. The lower excretion of 2-MeOE1 and 2-MeOE2 in the urine of endometrial cancer patients indicated that the methylation pathway may play an important role in the inhibition of endometrial cancer. The results from this study could help elucidate the association of the estrogen metabolic pathway with carcinogenesis and could also provide potential biomarkers for the assessment of estrogen-induced breast cancer risk. The newly developed method could help to practically aid the clinician in the diagnostic workup for endometrial cancer.
Acknowledgements
▼
This work was supported by the Hebei medical science research (number: 20100118) and by funds for the Construction of Potentially Distinguished Scientific Projects in the program of Hebei Universities. We would like to thank the Department of Gynecology of the 4th hospital and the Institute of Hebei Cancer Research. We are especially grateful to Ye Jiang and Yan Liu for their excellent technical assistance. We are also grateful to the patients and healthy controls for agreeing to participate in this study.
16aOHE1/ 2-OHE1
2-OHE1/ 2-MeOE1
Fig. 4 Relationship between parent estrogen and its metabolite. The specific value between estrogens and their metabolites in the urine were displayed in healthy women (Controls n = 23) and endometrial cancer patients (Cases n = 23). The distribution of date was unbalanced; the date was shown as the median. The statistics was performed by nonparametric test. *p < 0.05. E2: 17β-Estradiol; 4-OHE2: 4-Hydroxyestradiol; 2-OHE2: 2-Hydroxyestradiol; 2-MeOE2: 2-Methoxyestradiol; E1: Estrone; 16α-OHE1: 16α-Hydroxyestrone; 2-OHE1: 2-Hydroxyestrone; 2-MeOE1: 2-Methoxyestrone.
Conflict of Interest
▼
The authors declare that they have no conflicts of interest in the authorship or publication of this contribution.
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