Anal. Chem. 2007, 79, 3734-3740
Preparation of Antibodies for the Designer Steroid Tetrahydrogestrinone and Development of an Enzyme-Linked Immunosorbent Assay for Human Urine Analysis J.-Pablo Salvador, Francisco Sanchez-Baeza, and M.-Pilar Marco*
Applied Molecular Receptors group (AMRg), Department of Biological Organic Chemistry, IIQAB-CSIC, Jorge Girona 18-26, 08034-Barcelona, Spain
A highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed for tetrahydrogestrinone (THG), the new designer anabolic steroid responsible for the well-known Balco scandal announced in year 2003. Antibodies have been raised against 18a-homo-pregna4,9,11-trien-17β-ol-3-carboxymethyl oxime coupled to horseshoe crab hemocyanin. The hapten has been synthesized from gestrinone by controlled reduction of the triple bond of the ethinyl group at position C-17, without affecting the double bonds of the steroidal rings, followed by reaction of the keto group at C-3 with (carboxymethoxy)amine hemihydrochloride to form the oxime bond. The antisera obtained has been used in combination with 18a-homo-pregna-4,9,11-trien-17β-ol-20-yn-3-carboxymethyl oxime, a hapten derivative of gestrinone, coupled to bovine serum albumin to establish a competitive ELISA. Under the conditions used, THG can be detected in buffer with a limit of detection (LOD) of 0.045 ( 0.015 µg L-1 (N ) 9). The assay is very selective since other steroids assessed are not recognized. Preliminary experiments performed with human urine samples demonstrate that the assay can be applied to the analysis of these samples after a simple sample treatment method reaching a LOD of 0.25 ( 0.14 µg L-1. Accuracy is very good as demonstrated by the excellent correlation obtained when analyzing blind spiked urine samples (slope 0.93, R2 ) 0.992). In October 2003 the Food and Drug Administration was aware that an unapproved new drug called tetrahydrogestrinone (THG) was being used by athletes to improve their performance. THG was the second designer steroid chemically produced to be undetectable in the drug test being used at the time. Norbolethone was the first one, identified in 2002 by chemists of the Olympic Analytical Laboratory at the University of California (UCLA, Los Angeles). The use of THG was discovered thanks to a syringe sent to the U.S. Anti-Doping Agency by a coach noticing that it contained a substance widely used by athletes. The syringe was sent to one of the International Olympic Committee (IOC) testing * To whom correspondence should be addressed. Phone: +34 93 4006100. Fax: +34 93 2045904. E-mail:
[email protected].
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laboratories in UCLA, under the supervision of Prof. Catlin.1 After different GC/MS and LC/MS/MS studies, the structure of THG was elucidated2 as a new designer anabolic androgenic steroid (AAS) with a chemical structure similar to gestrinone and trenbolone. The substance resulted to be 18a-homo-pregna4,9,11-trien-17β-ol-3-one with proved activity over the androgen receptor (AR) and also with significant progestin activity.3 Once identified, in 2005 it was included in the list of banned substances for the World Anti-Doping Agency (WADA) and the IOC.4 The discovery of these AASs prompted concern that a range of novel androgens might be produced from marketed progestins and other synthetic sex steroids. In fact, in February 2005 WADA announced the discovery of the third designer steroid, desoxymethyltestosterone (DMT), thanks to an anonymous e-mail addressed to the agency to investigate a substance seized by Canadian customs. Since then, the Doping Control Laboratory at the University of Quebec in Montreal is presently conducting studies on DMT to identify metabolites and physiological properties of this steroid.5 While there are no reports regarding DMT use by athletes, THG has already been detected several times in recent athletic competitions. The scandal has damaged other sports such as baseball and American football. These data indicate that the use of this new drug among athletes could be quite frequent.6 Apart from obvious aspects of equality and fair play, the use of androgenic steroids holds associated risks inherent to their anabolic and androgenic properties. Hence, existing scientific data, which consist of case reports and clinical observations, describe serious cardiovascular adverse effects from use of performanceenhancing substances, including sudden death.7 Similarly, the effect of using androgenic steroids on the sperm quality has also (1) Ritter, S. K. Sci. Technol. 2003, 81, 66-69. (2) Catlin, D. H.; Sekera, M. H.; Ahrens, B. D.; Starcevic, B.; Chang, Y.-C.; Hatton, C. K. Rapid Commun. Mass Spectrom. 2004, 18, 1245-1249. (3) Death, A. K.; McGrath, K. C. Y.; Kazlauskas, R.; Handelsman, D. J. Obst. Gynecol. Surv. 2004, 59, 714-716. (4) WADA. http://www.wada-ama.org/en/dynamic.ch2?pageCategory_id)47 2005. (5) Ritter, S. Chem. Eng. News 2005, February 11 (updated February 24, 2005), Latest News. (6) Nicholls, H. Chem. World 2004, 1, 54-57. (7) Dhar, R.; Stout, C. W.; Link, M. S.; Homoud, M. K.; Weinstock, J.; Estes, N. A. M. Mayo Clinic Proc. 2005, 80, 1307-1315. 10.1021/ac061757x CCC: $37.00
© 2007 American Chemical Society Published on Web 04/18/2007
been demonstrated.8 Recently, Labrie et al.9 reported the results of their studies regarding the potential anabolic/androgenic activity of THG by identifying its effect on the expression of the mouse genome (near 30 000 genes) and with respect to the effect of dihydrotestosterone (DHT), the most potent natural androgen. Both steroids modulated the same genes in a similar manner. However, although under in vivo conditions THG possesses 20% of the potency of DHT in stimulating prostate, seminal vesicle, and certain muscles in mouse, THG was found more potent than DHT in binding to the AR. Therefore, there is a high risk for undesirable effects on the health status of individuals consuming this type of hormone. Detection of drug use is not a trivial analytical task. Before routine implementation, there are many questions to be answered such as the type of sample to be collected, identification of the proportion between the parent compound and the metabolites present in a particular type of sample, selection of an efficient extraction procedure, and a selective and sensitive detection method, etc. For drugs that have routine medical applications, information on the pharmacokinetic/pharmacodynamics is usually available. However, athletes often use nonapproved anabolic steroids or those approved only for veterinary applications. Since metabolism is different in animals and in man, human excretion needs to be determined. Regarding THG, no legitimate in vivo human excretion studies identifying urinary markers of this doping agent have been reported. The most reliable information available belongs to in vitro systems using human hepatocytes. According to these studies based on HPLC/MS/MS and NMR data, an in vitro metabolic pathway leading to the addition of a hydroxyl group followed by the β-glucuronic acid at C-18 has been proposed as the major metabolic route. Second, the formation of a glucoronide conjugate of an oxidative product with a hydroxyl group probably at C-16 has been suggested.10 The information reported from these in vitro experiments have provided the basis for further identification of THG human urinary markers. Meanwhile, analytical chromatographic methodologies have been established, aimed at detecting THG related substances,10-12 to perform these metabolic, biochemical, and activity studies. In an effort to discover new designer steroids early, Nielen et al.13 have recently reported the combination of a novel yeast reporter gene androgen bioassay with HPLC coupled to time-of flight mass spectrometry (LC/ QTOFMS) which allows accurate mass measurements of the bioassay positive results. Alternatively, the high-sample throughput capability of the immunochemical methods could respond to the demands of the control for the illegal use of this drug by elite amateur and professional athletes. It has been often demonstrated that immunoassays can provide the necessary reliability, low cost (8) Torres Calleja, J.; Gonzalez Unzaga, M.; DeCelis Carrillo, R.; Calzada Sanchez, L.; Pedron, N. Life Sci. 2001, 68, 1769-1774. (9) Labrie, F.; Luu-The, V.; Calvo, E.; Martel, C.; Cloutier, J.; Gauthier, S.; Belleau, P.; Morissette, J.; Levesque, M.-H.; Labrie, C. J. Endocrinol. 2005, 184, 427-433. (10) Levesque, J.-F.; Templeton, E.; Trimble, L.; Berthelette, C.; Chauret, N. Anal. Chem. 2005, 77, 3164-3172. (11) Karpiesiuk, W.; Lehner, A. F.; Hughes, C. G.; Tobin, T. Chromatographia 2004, 60, 359-363. (12) Thevis, M.; Geyer, H.; Mareck, U.; Schaenzer, W. J. Mass Spectrom. 2005, 40, 955-962. (13) Nielen, M. W. F.; Bovee, T. F. H.; van Engelen, M. C.; Rutgers, P.; Hamers, A. R. M.; van Rhijn, I. H. A.; Hoogenboom, L. Anal. Chem. 2006, 78, 424431.
of the analysis/sample, ease of use, selectivity, and detectability to analyze small organic molecules (i.e. refs 14 and 15). Thus, immunochemical analytical methods have proved to be appropriate tools for the screening other anabolic steroids used as doping agents or growth promoters in farm animals.16-18 Moreover, in this case availability of antibodies may help to identify other metabolites or bioconjugates structurally related with THG as well as to complete the THG ADMET (adsorption, distribution, metabolism, excretion, and toxicity) profile. Attending to these facts, this paper presents for the first time the preparation of antibodies and the necessary immunoreagents for THG determination. These immunoreagents can be the base for further development of bioanalytical methods based on different protocols (i.e., immunoaffinity extraction procedures, immunoassay, westernblot, etc.) and sensing principles (i.e., optical, electrochemical, or piezoelectric immunosensors) as well as other biochemical investigations. Here we present the characterization of the antibodies through the development of an ELISA protocol for the detection of THG. The assay has proven to be useful to analyze this substance in human urine samples. EXPERIMENTAL SECTION General Methods and Instruments. Thin layer chromatography was performed on 0.25 mm precoated silica gel 60 F254 aluminum sheets (Merck, Gibbstown, NJ), and the separations of the different compounds synthesized were done by column chromatography with silica 60 A C.C. 35-70 µm sodium dodecyl sulfate. 1H and 13C NMR spectra were obtained with a Varian Inova-500 (Varian Inc., Palo Alto, CA) spectrometer (500 MHz for 1H and 125 MHz for 13C). Infrared spectra were measured on a Bowmen MB120 FT-IR spectrophotometer (Hartmann & Braun, Que´bec, Canada). HPLC-UV analysis were performed using a Merck Hitachi L-7100 pump provided with a diode array L-7455 detector, a L-7200 autosampler, and a D7000 interface (Merck, Darmstadt, Germany). The chromatograms were processed with the HSM software (Merck, Darmstadt, Germany). The column used was Lichrospher 100 RP-18 125 × 4 (5 µm; Merck, Darmstadt, Germany), and the analyses were performed on isocratic mode using acetonitrile (ACN):H2O 6:4 as mobile phase at a flow rate of 1.0 mL min-1. The reactions were monitored at three wavelengths: 345, 310, and 254 nm. Preparative HPLC was performed using a Waters Prep LC4000 pump (Millipore Corp., Milford, MA) and a Waters Prepack 1000 pressure module with a flow distributor where 2% of the sample goes to the MerckHitachi L-4000 detector (Merck, Darmstadt, Germany). The column was a Perkin-Elmer Preparative C18 Flow, and the mobile phase was passed at a flow rate of 12 mL min-1 with the following (14) Estevez, M.-C.; Font, H.; Nichkova, M.; Salvador, J.-P.; Varela, B.; SanchezBaeza, F.; Marco, M.-P. In Emerging Organic Pollutants in Waste Waters and Sludge; Barcelo´, D., Ed.; Springer-Verlag: Berlin, 2005; Vol. 2, pp 119180. (15) Estevez, M.-C.; Font, H.; Nichkova, M.; Salvador, J.-P.; Varela, B.; SanchezBaeza, F.; Marco, M.-P. In Emerging Organic Pollutants in Waste Waters and Sludge; Barcelo´, D., Ed.; Springer-Verlag: Berlin, 2005; Vol. 2, pp 181244. (16) Gleixner, A. Fleischwirtschaft 1997, 77, 1108-1110. (17) Degand, G.; Maghuin-Rogister, G.; Delahaut, P.; De Bie, E.; Cox, C.; Havaux, J. C.; Techland, S. A. Food Saf. Qual. Assur.: Appl. Immunoassay Syst. 1992, roc., 1st, Meeting Date 1991, 1241-1993. (18) Scippo, M. L.; VandeWeerdt, C.; Willemsen, P.; Francois, J. M.; RentierDelrue, F.; Muller, M.; Martial, J. A.; MaghuinRogister, G. Anal. Chim. Acta 2002, 473, 135-141.
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Figure 1. Scheme showing the synthesis of haptens 3OCMO-THG and 3OCMO-G. 3OCMO-THG was covalently coupled to HCH and used to raise polyclonal antibodies. 3OCMO-THG and 3OCMO-G were coupled to BSA and used as coating antigens for ELISA development. The figure shows the usual way to name the steroid rings.
gradient program: min 0, ACN:H2O 53:47; min 5, ACN:H2O 53: 47; min 30, ACN:H2O 73:27. LC/ESI/MS (liquid chromatography/ electrospray ionization/mass spectrometry) was an Agilent 1100 series and was used in SCAN mode, monitoring positive ions, with simultaneous detection with a diode array detector. The column used in this case was Lichrospher 100 RP-18 125 × 4 (5 µm; Merck, Darmstadt, Germany), and the analyses were performed on isocratic mode using ACN:H2O 6:4 as mobile phase at a flow rate of 1.0 mL min-1. The MALDI-TOF-MS (matrix assisted laser desorption ionization time-of-flight mass spectrometer) used to characterize the protein conjugates was a Bruker Biflex III (Bruker, Kalsruhe, Germany) equipped with a laser unit that operates at a wavelength of 337 nm and the maximum output of 6 mW. The pH and the conductivity of all buffers and solutions were measured with a pH meter 540 GLP and a conductimeter LF 340, respectively (WTW, Weilheim, Germany). Polystyrene microtiter plates were purchased from Nunc (Maxisorp, Roskilde, Denmark). Washing steps were carried out using a SLY96 PW microplate washer (SLT Labinstruments GmbH, Salzburg, Austria). Absorbances were read using a SpectramaxPlus (Molecular Devices, Sunnyvale, CA) at a single wavelength mode of 450 nm. The competitive curves were analyzed with a four-parameter equation using the software SoftmaxPro v2.7 (Molecular Devices) and GraphPad Prism v 4 (GraphPad Software Inc., San Diego, CA). Unless otherwise indicated, data presented correspond to the average of at least two well replicates. Immunochemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Gestrinone was purchased from Sequoia Research Products, Ltd (Oxford, U.K.). Other chemical reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI). Buffers. PBS is 10 mM phosphate buffer 0.8% saline solution, and unless otherwise indicated the pH is 7.5. Borate buffer is 0.2 M boric acid/sodium borate pH ) 8.7. Coating buffer is 50 mM carbonate-bicarbonate buffer pH ) 9.6. PBST is PBS with 0.05% Tween 20. 2X PBST is PBST double-concentrated. Citrate buffer is a 40 mM solution of sodium citrate pH ) 5.5. The substrate 3736
Analytical Chemistry, Vol. 79, No. 10, May 15, 2007
solution contains 0.01% TMB (tetramethylbenzidine) and 0.004% H2O2 in citrate buffer. Hapten Synthesis. 18a-Homo-pregna-4,9,11-trien-17β-ol-3-carboxymethyl oxime: Hapten 3OCMO-THG. The THG was synthesized by hydrogenation of gestrinone (G) as described in the Supporting Information. (Carboxymethoxyl)amine hemihydrochloride (40.9 mg, 0.18 mmol) was added to a freshly prepared solution of THG (57 mg, 0.18 mmol) in 1 N KOH/EtOH (1 mL), and the mixture was kept under reflux for 1 h, until the complete disappearance of the starting material by TLC analysis. After the reaction was finished, the crude material was acidified with concentrated HCl and the EtOH removed under reduced pressure. The solid obtained was redissolved with ethyl acetate (AcOEt) (10 mL), washed with 1 N HCl (3 × 5 mL), and extracted with 1 N NaOH (3 × 5 mL). The aqueous layer was acidified with concentrated HCl and extracted with AcOEt (3 × 5 mL), and the organic layer was washed with water (2 × 5 mL), dried with anhydrous MgSO4, filtered, and evaporated to dryness under reduced pressure to obtain the desired hapten as a 1:1 mixture of Z and E isomers (38.7 mg, 55% of yield). 1H NMR (500 MHz, CDCl3) δ: 6.57 (1H, d, C11H, J ) 10.0 Hz), 6.29, 6.24 (1H, 2 × d, C12H, J ) 10.0 Hz), 6.46, 5.88 (1H, 2 × s, C4H), 4.62, 4.64 (2H, 2 × s, OCH2CO2), 2.73-2.46 (11H, m), 1.46-2.10 (7H, m), 1.06 (3H, t, C18bH3, J ) 7.69 Hz), 0.97 (3H, t, C20bH3, J ) 7.43 Hz). HRMS (+EI) calc for C23H31NO4 (M+) 385.225309, found 385.226913. 18a-Homo-pregna-4,9,11-trien-17β-ol-20-yn-3-carboxymethyl oxime: Hapten 3OCMO-G. Following the analogous strategy described above, gestrinone (G) (50 mg, 0.16 mmol) was reacted with (carboxymethoxy)amine hemihydrochloride (41.8 mg, 0.19 mmol) to obtain the desired competitor hapten as a 1:1 mixture of Z and E isomers (45.7 mg, 75% yield). 1H NMR (500 MHz, CDCl3) δ: 6.50 (1H, d, C11H, J ) 10.5 Hz), 6.26, 6.30 (1H, 2 × d, C12H, J ) 10.2 Hz), 6.42, 5.78 (1H, 2 × s, C4H), 4.52, 4.51 (2H, 2 × s, OCH2CO2), 2.79-1.18 (16H, m), 2.45 (1H, s, tCH20b), 0.93 (3H, t, C18bH3, J ) 7.69 Hz), 0.97 (3H, t, C20bH3, J ) 7.56 Hz).
Figure 2. Sequence of the hydrogenation reaction determined according to HPLC/UV and LC/ESI(+)/MS data. Under each structure is shown the λmax (HPLC/UV) and the [M + 1] (HPLC/MS) used to follow the reaction. The graph shows the kinetic of the hydrogenation reaction of gestrinone (G) to synthesize tetrahydrogestrinone (THG) using H2 at atmospheric pressure and Pd/BaSO4 as catalyst. G is reduced to form first dihydrogestrinone (DHG) and subsequently THG. After a certain time, the formation of other hydrogenation byproducts (HHG, hexahydrogestrinone; ROHG, R-octohydrogestrinone) is observed. The values were normalized from the data obtained of HPLC/UV. Table 1. Features of the Different ELISAs Obtained with the As168-170 and 3OCMO-THG-BSA and 3OCMO-G-BSA as Coating Antigens Ag
As
Amax
Amin
IC50, nM
IC50, µg L-1
slope
r2
3OCMO-THG-BSA 3OCMO-THG-BSA 3OCMO-THG-BSA 3OCMO-G-BSA 3OCMO-G-BSA 3OCMO-G-BSA
168 169 170 168 169 170
1.32 1.03 1.15 1.30 0.75 1.11
0.02 0.03 0.03 0.01 0.03 0.05
12.0 8.50 5.11 4.71 2.33 1.52
3.74 2.65 1.59 1.46 0.72 0.47
-0.93 -0.79 -0.70 -0.80 -0.79 -0.85
0.990 0.996 0.998 0.993 0.995 0.995
HRMS (+EI) calc for C23H27NO4 (M+) 381.194008, found 381.192417. Preparation of the Protein Conjugates. The haptens 3OCMOTHG and 3OCMO-G were coupled to HCH (horseshoe crab hemocyanin) and to BSA (bovine serum albumin) following the mixed anhydride method as previously described.19 Briefly, the carboxylic acids of 3OCMO-THG and 3OCMO-G (10 µmol) were activated with isobuthylchloroformate (14 µmol) in the presence (19) Galve, R.; Camps, F.; Sanchez-Baeza, F.; Marco, M.-P. Anal. Chem. 2000, 72, 2237-2246.
of tributhylamine (12 µmol, respectively) in DMF (dimethylformamide, 200 µL) and added to a solution of the protein (10 mg) in borate buffer. MALDI-TOF-MS spectra were obtained by mixing 2 µL of the matrix (trans-3,5-dimethoxy-4-hydroxycinnamic acid, 10 mg mL-1 in CH3CN/H2O 70:30, 0.1% TFA) with 2 µL of a solution of the conjugates or proteins (5 mg mL-1 in MilliQ water). The hapten densities obtained were around 6 and 7 for 3OCMOTHG-BSA and 3OCMO-G-BSA, respectively. Polyclonal Antisera. Three female New Zealand white rabbits weighing 1-2 kg were immunized with 3OCMO-THG-HCH Analytical Chemistry, Vol. 79, No. 10, May 15, 2007
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Figure 3. Effect of the ionic strength (A) and the pH (B) of the buffered media on the As170/3OCMO-G-BSA immunoassay features. The legends at the top of the left or right Y-axes indicate which parameters are represented. The IC50 is expressed in nM. Table 2. Features of the As170/3OCMO-G-BSA after Evaluationa Amax Amin IC50, µg L-1 slope dynamic range, µg L-1 LOD, µg L-1 R2
0.88 ( 0.14 0.03 ( 0.01 0.45 ( 0.14 -0.991 ( 0.14 0.108 ( 0.032 to 1.983 ( 0.713 0.045 ( 0.015 0.992 ( 0.004
a Values obtained correspond to the average and standard deviation of each parameter of nine assays performed in three different days.
Figure 4. Calibration curve of the As170/3OCMO-G-BSA immunoassay. The data shown in the graph are the averages and standard deviations of nine assays performed in three different days. Each concentration was tested using two well replicates on every assay. The parameters of the assay are shown in Table 2.
described.19
according to the immunization protocol previously Evolution of the antibody titer was assessed by measuring the binding of serial dilutions of the antisera to microtiter plates coated with the homologous BSA conjugate as described.20 After an acceptable antibody titer was observed, the animals were exsanguinated and the blood was collected in vacutainer tubes provided with a serum separation gel. Antiserum was obtained by centrifugation and stored at -40 °C in the presence of 0.02% NaN3. Indirect ELISA. The appropriate dilutions of the antisera and the concentration of the coating antigen were established after a two-dimensional checkerboard titration assay performed as described.20 Microtiter plates were coated with the antigen 3OCMOG-BSA (0.125 µg mL-1 in coating buffer 100 µL/well) overnight at 4 °C. The following day the plates were washed with PBST (4 × 300 µL), and solutions of the THG standards (from 1000 nM to 0.064 nM), cross-reactants (same concentration range), or samples prepared in 10 mM PBST (50 µL/well) were added followed by the solution of the antiserum As170 (1/16000 diluted in 10 mM PBST, 50 µL/well). After 30 min at room temperature, the plates were washed again as before and the anti-IgG-HRP solution (1/6000 in PBST, 100 µL/well) was added to the wells and incubated for 30 min more. After another cycle of washes, the substrate solution was added (100 µL/well), and the enzymatic reaction was stopped after 30 min at room temperature with 4 N H2SO4 (50 µL/well). The absorbances were measured at 450 nm. The standard curve was fitted to a four-parameter logistic equation (20) Este´vez, M.-C.; Kreuzer, M.; Sa´nchez-Baeza, F.; Marco, M.-P. Environ. Sci. Technol. 2006, 40, 559-568.
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Table 3. Cross-Reactivity of Related Steroidal Compounds in the As170/3OCMO-G-BSA ELISA compound
IC50, nM
% CR
tetrahydrogestrinone gestrinone norethandrolone trenbolone androstenedione androstan-diene-dione stanozolol dihydrotestosterone boldenone methylboldenone testosterone progesterone pregnenolone ethynylestradiol estrone cholesterol
1.61 25.75 7.96 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000
100 6 20
