Steroids 78 (2013) 297–303
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Steroids
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Discriminating the endogenous and exogenous urinary estrogens in human by isotopic ratio mass spectrometry and its potential clinical value ⇑ Sheng Yang a, , Dapeng Zhang b, Youxuan Xu a, Xiaobing Wang a, Xin Liu a, Shan Wang a, Jingzhu Wang a, Moutian Wu a, Zhenwen He a, Jian Zhao a, Hong Yuan a a National Laboratory of Anti-Doping, China Anti-Doping Agency, Beijing 100029, China b Neixiang Center for Disease Control and Prevention, Henan Province 474350, China article info abstract
Article history: Estrogens were prohibited in the food producing animals by European Union (96/22/EC directive) and Received 18 May 2012 added to the Report on Carcinogens in United States since 2002. Due to very low concentration in serum Received in revised form 17 October 2012 or urine ( pg/mL), the method of control its abuse had not been fully developed. Accepted 17 November 2012 The endogenous estrogens were separated from urines of 18 adult men and women. The exogenous Available online 7 December 2012 estrogens were chemical reference standards and over the counter preparations. Two patients of dysfunc- tional uterine bleeding (DUB) administered exogenous estradiol and the urines were collected for 72 h. Keywords: The urinary estrogens were separated by high-performance liquid chromatography (HPLC) and con- Drug abuse firmed. The exogenous and exogenous estrogens were analyzed by gas chromatography combustion iso- Estrogen 13 12 13 Endogenous tope ratio mass spectrometry (GC–C–IRMS) to determine the C/ C ratio (d C‰). 13 Exogenous The d C‰ values of reference standard of E1, E2, and E3 were 29.36 ± 0.72, 27.98 ± 0.35, 13 Urine 27.62 ± 0.51, respectively. The d C‰ values of the endogenous E1, E2, and E3 were 21.62 ± 1.07, Gas chromatography combustion isotope 22.14 ± 0.98, and 21.88 ± 1.16, with P < 0.01 (t-test). Two DUB patients’ urinary estradiol d13C‰ values ratio mass spectrometry was depleted to 28.02 ± 0.33 after the administration. The progesterone, 17a-hydroxyprogesterone, pregnanediol, as well as desogestrel and ethinylestradiol from contraceptives were also determined. Stable carbon isotope analysis can distinguish the endogenous and exogenous urinary estrogen in human. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction one, E1), 17b-estradiol (estra-1,3,5(l0)-triene-3,17b-diol, E2), and estriol (estra-1,3,5(l0)-triene-3,16a,l7b-triol, E3), respectively, Estrogens are naturally synthesized in the ovaries, adrenal Fig. 1 [1,2]. Estrogens play an important role in the estrous cycle glands, and metabolized in the liver in human. The three major [3]. In female, estrogen levels vary through the menstrual cycle natural estrogens are estrone (3-hydroxyestra-1,3,5(10)-trien-17- [4], with levels highest just before ovulation. They promote the development of female secondary sexual characteristics, such as
Abbreviations: A, androsterone; amu, atomic units; CIR, carbon isotopic ratio; breasts. In male, estrogen regulates the maturation of sperm [5]. C.V.%, coefficient of variance; DUB, dysfunctional uterine bleeding; E1, estrone; E2, Estradiol and estrone can be converted from testosterone and 17b-estradiol; E3, estriol; Etio, etiocholanolone; eV, electron volt; GC–C–IRMS, gas androstenedione; the conversion is catalyzed by enzyme aroma- chromatography combustion isotope ratio mass spectrometry; GC–MS, gas chro- tase (EC 1.14.14.1) [6,7]. matography mass spectrometry; GnRH, gonadotropin-releasing hormone; HCG, Due to their important physiological effects, estrogens are human chorionic gonadotropin; Hgb, hemoglobin; HPLC, high-performance liquid chromatography; HRT, hormone replacement therapy; 125I, iodine element (125); strictly prescribed to the treatment of dysfunctional uterine bleed- I.D., inner diameter; IRMS, isotopic ratio mass spectrometry; LC–MS, liquid ing (DUB) [8,9], menopause, and in the hormone replacement ther- chromatography mass spectrometry; LOD, limit of detection; m/z, mass to charge apy (HRT) [10] in gynecological clinics. However, as a major ratio; MTBE, methyl tert-butyl ether; MSTFA, N-Methyl-N-(trimethylsilyl) trifluo- component in the contraceptive pills, estrogens can be easily ob- roacetamide; NIEHS, National Institute of Environmental Health Sciences; PD, pregnanediol; QC, quality control; RIA, radioimmunoassay; SD, standard deviation; tained over the counter. According to an epidemiological survey SPSS, Statistical Package for the Social Sciences; VPDB, Vienna Pee Dee Belemnite; in Fujian province (34.8 million population), southeast China, there WADA, World Anti-Doping Agency. were 78% children (n = 80) diagnosed of precocious puberty had a ⇑ Corresponding author. Address: China Anti-Doping Agency, No. 1, Anding Road, history exposure to exogenous sexual hormone [11]. The gonado- Chaoyang District, Beijing 100029, China. Tel.: +86 10 64946933; fax: +86 10 tropin-releasing hormone (GnRH) stimulating trial had to be per- 64979303. E-mail address: [email protected] (S. Yang). formed to clarify the etiology, but sometimes it was refused by
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.steroids.2012.11.013 298 S. Yang et al. / Steroids 78 (2013) 297–303
O CH OH OH 3 CH3 CH3
H H H OH
H H H HHH HO HO HO 3 1 2
OH O O CH CH3 CH3 3 CH CH3 3 CH3 OH CH3 H CH CH3 H 3 H
H H H H H H O O HO H 6 4 5
OH CH OH OH 3 CH CH3 CH CH 3 CH H2C H H H H H
H H H H H H O HO 8 7 9
Fig. 1. The structure of estrone, 1; estradiol, 2; 17b-estriol, 3; progesterone, 4; 17a-hydroxyprogesterone, 5; pregnanediol, 6; norethisterone, 7; desogestrel, 8; ethinylestradiol, 9. parents for the considerations of worsen the condition. If the uri- 2. Experimental nary estrogens can be detected and indicated the sources, namely synthesized within the organism (endogenous) or from the outside 2.1. Chemicals (exogenous), the differential diagnosis would be relative safer and easier. In oncology, about 75% of breast cancers are known as hor- Estrone (3-hydroxyestra-1,3,5(10)-trien-17-one), estradiol (es- mone-sensitive or estrogen-receptor-positive cancers [12].In tra-1,3,5(l0)-triene-3,17b-diol), estriol (estra-1,3,5(l0)-triene- European Union, anabolic agents including estrogen were prohib- 3,16a,l7b-triol), progesterone (pregn-4-ene-3,20-dione), 17a- ited in food producing animals (96/22/EC directive). And in the hydroxyprogesterone (4-pregnen-17a-ol-3,20-dione), and preg- United States, estrogen had been added to the Report on Carcino- nanediol (5b-pregnane-3a,20a-diol) were chemical references gens by National Institute of Environmental Health Sciences (NIE- from Sigma–Aldrich. The 19-norethisterone (17a-ethynyl-17b-hy- HS) since the year 2002 [13]. If the preventive measures can be droxy-19-nor-4-androsten-3-one) was obtained from national performed in the food safety area, nutritional supplements prod- institutes for food and drug control of China. The contraceptive ucts, and cosmetics as well, the incidence of breast cancer may drugs of YasminÒ, MarvelonÒ, and MercilonÒ were purchased from be lowered. Therefore, it is necessary to develop a method to con- the pharmacy in Beijing. Testosterone and androsterone obtained trol the estrogens abuse. from Sigma–Aldrich, which were used as external standards for The earlier published method for quantifying estrogens had HPLC and GC–C–IRMS analysis. Methyl tert-butyl ether (MTBE) used radioimmunoassay (RIA) [14]. This technique had the limita- and acetonitrile were of HPLC grade obtained from Dima. Analyti- tions that radiological 125I was used as a tracer, and the antibody cal grade of hydrochloric acid, sodium dihydrogen phosphate had cross-reactivity with other substances. Although estrogens monohydrate, and disodium hydrogen phosphate dehydrate were have been determined at trace levels (ng/L) by mass spectrometric purchased from Sinopharm Chemical Reagent. The N-Methyl-N- methods in human serum [15], river water [16], and sewage sedi- (trimethylsilyl) trifluoroacetamide (MSTFA), ammonium iodide ments [17]; these results could not provide sufficient information (NH4I), and dithioerythritol were from Sigma–Aldrich. The b-glu- about the origins of estrogens. The isotopic ratio mass spectrome- curonidase from Escherichia coli (G7396) and sulfatase from Helix try (IRMS) has been introduced in the anti-doping fields to control pomatia (S9626) were obtained from Sigma–Aldrich. Pure water testosterone abuse [18], an endogenous steroid. And one publica- was made by a Milli-Q system (Millipore). tion reported the findings in cattle after administration of exoge- nous estrogen by this technique [19]. We hypothesized the stable carbon isotopic ratio (CIR) is different between endogenous and 2.2. Individuals providing urine for estrogens isolation exogenous estrogens and asked whether this difference can be dis- tinguished by the IRMS methods. Our study investigated the car- Urine samples were collected from 18 healthy adults, including bon isotopic ratios of chemical reference estrogens and the 8 females (19–21 years old) and 8 males (26–45 years old) as well endogenous estrogens from 8 healthy subjects, as well as two cases as 2 pregnant women. For females, urines were collected during of estrogen administration (DUB) using the developed methods. the menstrual cycle, from the 9th day to the 15th day, for a phys- S. Yang et al. / Steroids 78 (2013) 297–303 299 iological higher concentration of estrogens. And 2 pregnant women constant flow mode) was helium. The oven temperature program enrolled provided urines for 2 weeks in the mid-term pregnancy was: 180 °C (2 min) 6 °C/min ? 255 °C 3 °C/min ? 285 °C (the 20th to the 22nd weeks). In males, urines were collected con- (6.5 min). The fractions were injected in split mode (1:10) at tinuously for 2 weeks. There was no contraceptive drug history be- 260 °C. Ion source used electron impact (EI) mode with 70 electron fore 1 month for all female volunteers. The diet was provided by a voltage (eV) at 230 °C. The full scan range was from 45 to university restaurant and not controlled as usual. Urine was pooled 450 atomic units (amu) with the scan rate of 4 scans per second. Ò in glass bottle (DURAN , 1000 mL) and frozen to 20 °C before For the derivatized estrogen (MSTFA/NH4I/dithioerythritol, analysis. For each subject, total of about 4.5 and 8.0 L for female 1000:2:3, v:w:w, at 70 °C for 30 min), the confirmation was per- and male respectively were extracted for determination endoge- formed on another Agilent 6890-5973 GC–MS with an Agilent nous 13C/12C ratio. Two patients (31 and 32 years) diagnosed of J&W HP-1 column (17 m 0.20 mm I.D., 0.11 lm film). The tem- dysfunctional uterine bleeding provided all the urines over the per- perature program was the same as mentioned above. The retention iod of 72 h after the estradiol administration for 3 days (3 mg twice time of bis (trimethylsilyl) estradiol was 13.40 min, with ions m/z a day). The study ensured that patients comply with the require- of 416, 326, 285, 232, and 129. ments of Helsinki Declaration. The study was evaluated and ap- proved by the institutional board of China Anti-Doping Agency. All the individuals signed a written consent. 2.5. The IRMS determination
2.3. Samples preparation and HPLC separation The carbon isotope ratio measurements were performed using a Delta–V Advantage isotope ratio mass spectrometer (Thermo Elec- Chemical reference standards (1.0 mg) were weighed by an tron). The GC–C–IRMS temperature program was set identical with electronic balance scale (Mettler), and dissolved by 1.0 mL metha- the GC–MS confirmation previously described to ensure all the nol in vials. A series of dilutions were prepared with the final con- analytes had the same retention time. The fractions were injected centrations of 100.0 ng/mL and used for quality controls. The in splitless mode at 260 °C. The combustion and reduction oven contraceptive preparations were dissolved by methanol (5 mL) in temperatures were set at 960 and 600 °C, respectively. Ion source glass tubes and vortexed for 5 min. After centrifugation (3000 g, energy was 99 eV. An ISODAT software (Version 2.5, Thermo Elec- 5 min), the upper phase was transferred in a fresh glass tube and tron) was installed on the computer. evaporated under nitrogen gas. Repeat the extraction for four times Ions (m/z 44, 45, and 46) were separated on a magnet and de- and collect the extracts for preparation reference solutions. tected by three Faraday collectors. Carbon isotope ratios of each The extraction methods adapted from Buisson et al. [19] and compound were expressed against the international standard modified by us. In short, urines (1000 mL) were aliquoted once in Vienna Pee Dee Belemnite (VPDB) [21,22]. 40 50 mL CorningÒ centrifuge tubes (Acton), with 25 mL urine in each tube. Urine samples (25 mL) were adjusted to pH 6.8–7.2 13 12 13 12 13 ð C= CÞsample ð C= CÞstd by the addition of phosphate buffer (0.8 mol/L, 10.0 ml, Na2HPO4:- d C‰ ¼ 13 12 1000 ð1Þ ð C= CÞstd NaH2PO4, 1:2, w:w). And the pH value was determined using an acidity meter. The b-glucuronidase (30 K unit) and sulfatase The calibration of the reference gas was performed with n-al- 13 (3000 U) were added and kept for 1.5 h at 55 °C. After cooling to kanes (C15 and C25) which d C VPDB-values were previously ambient temperature, urine samples (25 mL) were adjusted to calibrated. pH 5.0–5.5 by the addition of hydrochloric acid (11.9 mol/L) using a pipette. The liquid–liquid extraction used 15 mL MTBE, and the mixture shaken mechanically for 10 min, and subsequently centri- 2.6. Statistics fuged for 5 min (3000 g). The upper organic layer was transferred to a fresh glass tube, evaporated to dryness by nitrogen gas at The statistics was performed by SPSS software. The data are ex- 45 °C, and the dry residue was dissolved by 500 lL methanol. After pressed by mean ± standard deviations. The d13C values for the filtered by 0.2 lm membrane and transferred to a vial, volume of chemical reference estrogens were compared with the endogenous 80 lL for five times was fractionated with autosampler by HPLC. estrogens isolated from urine samples, using the general linear The clean up by HPLC was adapted from Wang et al. [20] and model. The difference between endogenous and exogenous estro- adjusted accordingly. The HPLC system was Waters 2796 biosepa- gens was tested by Student’s t-test, two tailed. rations module (Waters) with 2966 photodiode array detector (190–450 nm). A computer installed a MassLynx™ software was used. Separation was performed using an Agilent Zorbax SB-C18 2.7. Method validation column (250 mm 4.6 I.D., 5 lm particle size) at 25 °C. The mobile phase was acetonitrile and water (30:70, v:v), and the gradient in- To determine the ability to recover estradiol from urine, three creased from 30% to 100% in 18.0 min with flow rate at 1 mL/min. samples (3.0 L) of known concentrations were spiked with 3 levels The eluting fractions were collected in glass tubes by hand or by a low: 50 ng/L (185 pmol/L), medium: 100 ng/L (370 pmol/L), and fraction collector (Waters Fraction Collector III) and dried under high: 200 ng/L (740 pmol/L) of estradiol. The recovery was calcu- nitrogen gas. The fractional tubes were labeled with a marker lated by dividing the spiked amount by the expected concentration pen. The same fractions of multiple samples were dissolved by by GC–MS. The mean recoveries were 68.0%, 86.0% and 102.0% for MTBE and pooled for GC–MS confirmation and GC–C–IRMS low, medium and high levels, respectively. An external standard determination. reference solution of testosterone 500.0 ng/mL (1.78 nmol/L); 5 lL was injected on the HPLC to check the retention time 2.4. The fractions confirmed by GC–MS (11.32 ± 0.1 min). The coefficient of variance (C.V.%) of intra and in- ter-batch was less than 10.0%. The d13C‰ recorded in the experi- The analysis was performed on an Agilent 6890-5973 GC–MS. A ment with a prerequisite when its response (mV) value was Hewlett Packard computer with MSD chemstation software was higher than 100 (mV). The limit of detection (LOD) of GC–C–IRMS used. An Agilent HP-5 column (33 m 0.25 mm I.D., 0.33 lm film was 150.0 ng/L (550 pmol/L) estrogens (E1, E2, and E3) in urine. thickness) was used for separation. The carrier gas (1.2 mL/min, The lowest absolute loading quantity was 40 ng on column. 300 S. Yang et al. / Steroids 78 (2013) 297–303
3. Results radiol (17a-pregnatrien-20-yne-3,17-diol) in three preparations. The chromatograms of isotopic ratio mass spectrometry of desog- 3.1. Urine estrogens isolated by HPLC estrel was shown in Supplementary file (Supplementary Fig. 9).
An HPLC chromatogram showed the urinary target compounds 3.4. Case study I spiked in the urine; the retention times of each compound were determined and the eluting fraction was collected and identified Two Chinese women, 31 and 32 years old, were admitted into a by GC–MS. The collecting time range of E1 (10.5–11.5 min), E2 county hospital for prolongation menses with heavier bleeding. (8.8–10.2 min), and E3 (4.2–5.2 min) was selected. The corre- Physical exams were performed and homological analysis showed sponding time range were set by MassLynx software and used by Hgb 9.5 g/dL (1.47 mmol/L) and 11.0 g/dL (1.71 mmol/L); urine the Waters collector for the fractionation of urine. Besides estro- HCG tests were negative, and other parameters were at normal gens, progesterone and its metabolites were also determined, as ranges. Gynecological and B-ultrasonic examinations were per- well as the main component of contraceptive preparations. The formed to exclude pregnancy, trauma and tumor. The diagnosis monitoring windows of HPLC of relating substances will be helpful was dysfunctional uterine bleeding. And estradiol was prescribed to indicate the administration of contraceptive medicines, Fig. 2. and administered as ordered. The urines collected from two DUB patients were prepared by the previously developed methods. 3.2. GC–MS identification of urinary estrogens And the estradiol was confirmed by GC–MS and thereafter deter- mined by GC–C–IRMS. The CIR of estradiol was 28.02 ± 0.33, Urine estrogens (E1, E2, and E3) were dissolved in methanol, which was more depleted than the endogenous estradiol underivatized and identified by GC–MS. The molecular ions of E1, ( 22.32 ± 0.93, P < 0.01, t-test). E2, and E3 were 270, 272, and 288, respectively, which was consis- tent with the theoretical molecular weight. The abundances of 3.5. Case study II main fragments of nine compounds were summarized in Table 1. The mass spectra of estradiol and main fragments were explained, In nutritional supplements and cosmetic markets, there were Fig. 3. The mass spectra of estrone, estriol, progesterone, pregnane- some breast enlargement products claimed that they contain herbs diol, 17a-OH progesterone, 19-norethisterone, desogestrel, ethiny- with phyto-estrogenic properties. We screened 10 products ob- lestradiol were shown in the Supplementary file (Supplementary tained from the internet websites using the method of this paper. Figs. 1–8). One preparation claimed containing isoflavone but was confirmed exogenous estradiol. The mass spectrum of Bis (trimethylsilyl) 3.3. The carbon isotopic ratio of endogenous and exogenous estrogens estradiol and standard mass spectrum of NIST05 were displayed in the Supplementary file (Supplementary Fig. 10). The endogenous estrogens separated from urine were deter- mined by GC–C–IRMS. The retention times of nine compounds 4. Discussion were identical with the GC–MS identification. The d13C‰ of estro- gens were listed in Table 2. The mean CIR values of endogenous The estriol, estrone, and estradiol were isolated from pregnancy estrogens were 21.62 ± 1.07‰ (E1), 22.14 ± 0.98‰ (E2), and urine in the 1930s [1,2]. Hundreds of gallons of pregnancy urine 21.88 ± 1.16‰ (E3); the exogenous values were 29.36 ± 0.72‰ were distilled; theelin (estrone), theelol (estriol), and estradiol (E1), 27.98 ± 0.35‰ (E2) and 27.62 ± 0.51‰ (E3). The exogenous were isolated at microgram level. The concentrations of estrogens estrogens were more depleted than the endogenous estrogens in urine are estimated to be hundreds of nanograms per gallon. At (P < 0.01, t-test). The differences were larger than 5.0 per mil. present, using a semi-preparative HPLC and a C column can re- And this could be used as the criteria in the discriminating exoge- 18 cover hundreds of nanograms estrogens from 4 to 6 L urine. The nous estrogens. confirmation by GC–MS or LC–MS only needs 20–40 ng. The GC– The CIR of desogestrel (13-ethyl-11-methylene-18, 19-dinor- C–IRMS has the detection thresholds of 40 ng of estrogens. There- 17a-4-pregnen-20-yn-17-ol) from MarvelonÒ was fore, the endogenous estrogens can be determined by the devel- 33.81 ± 0.95‰ and that from MercilonÒ was 33.12 ± 0.66‰ oped methods. (P > 0.05, t-test). There was no difference for the CIR of ethinylest- The CIRs of other endogenous androgens, such as androsterone (3a-hydroxy-5a-androstan-17-one, A) and etiocholanolone (3a- hydroxy-5b-androstan-17-one, Etio) have been reported [23]. The reference range of A was 24.02‰ to 20.60‰, and Etio was 25.11‰ to 21.57‰ based on a reference population of 56 sub- jects. In human body, the conversion of testosterone to estradiol, and of androstenedione to estrone, is catalyzed by the enzyme aro- matase (EC 1.14.14.1) [24]. The catalytic process from testosterone to E2 does not alter the backbone structure of gonane. Hence, the composition of 13C/12C does not change significantly; we deduce that the endogenous estrogens have the similar CIR values as tes- tosterone and androstenedione. And the endogenous testosterones from 8 female subjects have been determined ( 24.68‰ to 19.41‰) by our laboratory, the results showed similar ranges as estrogens. The exogenous chemical references of estrogens, progesterone, and contraceptive preparations showed more depleted delta values than endogenous ones. At present, no report available can compare Fig. 2. Chromatogram of HPLC; A QC urine extracted and separated by Agilent with our findings. However, one study reported the CIR of seized Zorbax SB-C18; 17a-OH-P for 17a-hydroxyprogesterone, P for progesterone, Nor for 19-norethisterone. testosterone preparations have the range of 32.6‰ to 22.9‰, S. Yang et al. / Steroids 78 (2013) 297–303 301
Table 1 Characteristic fragment ions of compounds using GC–MS.
Compound Experiment [M+] Main fragments m/z (abundance %) Estrone EI full scan 270(100) 146(45) 185(40) 213(23) 242(4) Estradiol EI full scan 272(100) 213(44) 160(38) 186(16) 244(4) Estriol EI full scan 288(100) 146(45) 160(43) 213(30) 172(22) Progesterone EI full scan 314(29) 124(65) 272(26) 229(23) 299(5) Pregnanediol EI full scan 302(25)a 216(100) 234(77) 162(38) 269(18) 17a-Hydroxy progesterone EI full scan 330(22) 287(55) 229(43) 145(20) 269(15) Norethisterone EI full scan 298(31) 91(100) 231(43) 215(29) 270(7) Desogestrel EI full scan 310(12) 91(100) 213(23) 263(15) 281(13) Ethinylestradiol EI full scan 296(30) 213(100) 160(43) 133(28) 185(7)
a +Å Pregnanediol ion m/z of 302 was formed by [M –H2O].
Abundance
Scan 1949 (20.895 min): 12022310.D (-) 272
95
90 213(214-H)
85 OH 160 CH3 80 146 75
70
65
60 HO 55
50
45 213
40 160
35
30
133 25
20 186 115 15 91 10 41 67 5 244
293 310 327 343 359 375 391 0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z-->
Fig. 3. The mass spectra full scan of 17b-estradiol. with mean of 28.4‰, median of 28.6‰ [25]. It indicated that lestradiol, the distinctions would be obvious and relatively easier exogenous testosterone preparations have the similar ranges as by the comparison of their mass spectra (GC–MS or LC–MS). the exogenous estrogens and preparations; the underlying causes Although estrogens were not classified as doping agents, but its might be similar synthetic procedure in pharmaceutical manufac- prohormone (testosterone and androstenedione), aromatase inhib- turers. In general, the pharmaceutical preparations used phytoster- itors (such as aminoglutethimide, exemestane, and formestane), as ols as the starting materials extracted from soy or yams by partial well as the selective estrogen receptor modulators (raloxifene, synthesis [26]. In human, the endogenous steroids are synthesized tamoxifen, etc.) were all banned by World Anti-Doping Agency from cholesterol; the de novo synthesis of cholesterol is from acet- (WADA) [30]. Theses metabolic relations to estrogens were so close yl-coenzyme A, which the carbon sources can attribute to C3 [27] that could not be neglected. There were some publications indi- and C4 plants [28]. The distinctions between endogenous and exog- cated that pregnenolone (prohormone of pregesterone) can influ- enous steroids (same compound) were caused by isotopic compo- ence the CIR of steroid profile, which may be used as a masking sition of carbon in different substances. The IRMS take the agent, therefore suggest WADA to prohibit its use [31–33] in advantage of differential distribution of CIR in endogenous or exog- sports. And this prompted us to include the progesterone and its enous steroids [29]. Some exogenous estrogens, with different metabolites in this study. We met one urine sample in the routine molecular structures compared with E1, E2, and E3, such as ethiny- doping control years ago, and the athlete claimed in the urine sam- 302 S. Yang et al. / Steroids 78 (2013) 297–303
Table 2 The d13C‰ values of endogenous estrogens, chemical reference, and preparations.
Compound Sources/manufacturer Formula M.W. d13C‰ 13C Content % Determined Calculated Mean ± SD Mean
Estrone Human urine C18H22O2 270.37 21.62 ± 1.07 1.14801
17b-Estradiol Human urine C18H24O2 272.37 22.14 ± 0.98 1.14860
Estriol Human urine C18H24O3 288.38 21.88 ± 1.16 1.14831
Estrone Sigma–Aldrich C18H22O2 270.37 29.36 ± 0.72 1.15671
17b-Estradiol Sigma–Aldrich C18H24O2 272.37 27.98 ± 0.35 1.15516
Estriol Sigma–Aldrich C18H24O3 288.38 27.62 ± 0.51 1.15476
Progesterone Sigma–Aldrich C21H30O2 314.47 29.77 ± 0.48 1.15717
Pregnanediol Sigma–Aldrich C21H36O2 320.51 31.05 ± 0.81 1.15861
17a-Hydroxy-progesterone Sigma–Aldrich C21H30O3 330.46 29.51 ± 0.78 1.15688
19-Norethisterone Sigma–Aldrich C20H26O2 298.42 29.67 ± 0.65 1.15706 Ò Ethinylestradiol from Yasmin Schering C20H24O2 296.40 30.12 ± 0.52 1.15757 Ò Desogestrel from Marvelon N.V. Organon C22H30O 310.47 33.81 ± 0.95 1.16171 Ò Ethinylestradiol from Marvelon N.V. Organon C20H24O2 296.40 29.36 ± 0.28 1.15671 Ò Desogestrel from Mercilon Organon Ireland C22H30O 310.47 33.12 ± 0.66 1.16093 Ò Ethinylestradiol from Mercilon Organon Ireland C20H24O2 296.40 29.65 ± 0.74 1.15704 ple collection form that she took contraceptive pills for the purpose In conclusion, our results showed that the carbon isotopic ratios of adjustment of menstrual cycle, to prolonging the bleeding days of endogenous estrogens from humans were different with the to mismatch the competition. However, the IRMS result showed exogenous chemical references. And the GC–C–IRMS method could that A and Etio both exceed 28.0, which had reached the criteria distinguish the endogenous and exogenous estrogens. Moreover, of exogenous testosterone positive. In the following investigation, exogenous administered estrogens could be detected from the ur- the contraceptive preparations contained progesterone and ine by the method. estrogens. Therefore, there was the necessity of establishment a method to prevent the doping control laboratory to report a false Acknowledgments positive case. In addition, it is suggested that WADA should make clarify in its prohibited list whether the progesterone or pregnen- The authors are grateful to the subjects and patients in the olone is doping agent or not, because these substances could study and are grateful to China Anti-Doping Agency for providing metabolite to testosterone and estrogen; and giving explanations the instruments to fulfill the study. The help of engineers from Agi- of the ranges of ‘‘...other substances with a similar chemical struc- lent, Waters, and Thermo Fisher is greatly acknowledged. ture or similar biological effect(s).’’ in the end of the anabolic androgenic steroids. The developed method not only solved the problem of intake exogenous estrogens and/or progestins which Appendix A. Supplementary data might potentially cause a doping violation case by the athlete but also could be used in the investigation the reasons of some Supplementary data associated with this article can be found, in atypical results occurred in the IRMS analysis in anti-doping the online version, at http://dx.doi.org/10.1016/ practices. j.steroids.2012.11.013. In spite of estrogen is essential for the normal functioning of a woman’s reproductive system and for normal breast development References but long-term exposure to estrogen is thought to increase the risks of breast cancer. 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