RADIOIMMUNOASSAY OF 19 NOR TESTOSTERONE EVIDENCE OF ITS SECRETION BY THE TESTIS OF THE STALLION

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1 RADIOIMMUNOASSAY OF 19 NOR TESTOSTERONE EVIDENCE OF ITS SECRETION BY THE TESTIS OF THE STALLION E. Benoit, F. Garnier, D. Courtot, P. Delatour To cite this version: E. Benoit, F. Garnier, D. Courtot, P. Delatour. RADIOIMMUNOASSAY OF 19 NOR TESTOS- TERONE EVIDENCE OF ITS SECRETION BY THE TESTIS OF THE STALLION. Annales de Recherches Vétérinaires, INRA Editions, 1985, 16 (4), pp <hal > HAL Id: hal Submitted on 1 Jan 1985 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 RADIOIMMUNOASSAY OF 19 NOR TESTOSTERONE EVIDENCE OF ITS SECRETION BY THE TESTIS OF THE STALLION E. BENOIT F. GARNIER D. 2COURTOT P. DELATOUR 1 1: Laboratoire lnra n , Service de Biochimie, École Nationale Vétérinaire de Lyon, BP31, Charbonnières Cedex, France. 2: Laboratoire des Xénobiotiques chez le Cheval de Sport-lNRA, École Nationale Vétérinaire de Lyon, BP31, Charbonnières Cedex, France. Résumé MISE AU POINT D UN DOSAGE RADIO-IMMUNOLOGIQUE DE LA 19 NOR TESTOSTÉRONE. MISE EN ÉVIDENCE DE SA SÉCRÉTION PAR LE TESTICULE DE L ÉTALON. &horbar; Un antisérum a été produit chez le lapin à partir d un 1 70-hémisuccinale de 19 nor testostérone et utilisé pour la mise au point et la validation d un dosage radio-immunologique spécifique de la 19 nor testostérone plasmatique. Les différences de concentrations plasmatiques en 19 nor testostérone, lorsque les prélèvements sont effectués dans la veine jugulaire et testiculaire au cours de castration sous anesthésie générale permettent d affirmer l existence d une sécrétion testiculaire de 19 nor testostérone. De plus chez certains animaux cette sécrétion est inhibée corrélativement à la sécrétion de testostérone par le stress. The 19 nor testosterone or 1 7p-hydroxy-4-estren- 3-one (19 NT) was first synthetised under the name Nandrolone in It is used as an anabolic steroid and specially in competition horses. It was then thought to be only a xenobiotic. The urinary excretion by the stallion of 19 NT of endogenic origin was recently demonstrated by CG-MS (Courtot et al., 1984!. The existence of relatively low plasmatic concentrations of 19 NT compared to the urinary concentration, together with the necessity for a faster dosage method, made the development of a radio-immuno-assay (RIA) indispensable. The production of antibodies anti-19 NT was previously described by Jondorf (1977) as well as by Hampl and Picha (1 979! in order to study the pharmacokinetics of 19 NT administered as an anabolic agent. The low specificity of these antibodies against 19 NT (about 25 % and 6 % of cross reaction with testosterone, respectively) being of no consequent r pharmacological studies, makes the use of these antibodies particularly difficult in the evaluation of plasmatic endogenic 19 NT. This report concerns the production of specific antibodies anti-19 NT and the development of a RIA for plasmatic 19 NT evaluation. This assay is then used to demonstrate the secretion of this steroid by the testis of stallions. Materials and Methods 1. Preparation of the antiserum Antibodies were raised in rabbits treated with a 19 NT 17-hemisuccinate-bovine serum albumin conjugate. a) Preparation of 19 NT 17-hemisuccinate 19 NT (410 mg) and succinic anhydride (500 mg) dissolved in dry pyridine (5 ml) were refluxed for 4 hours (Erlanger et al., The product is then purified and cristallized fror acetone, benzene-hexane and again acetone. The structure of 19 NT 1 7-hemisuccinate

3 is ( E = confirmed by NMR and UV: Lmax 249 nm b) Coupling to bovine serum albumin The 19 NT 17-hemisuccinate was coupled to BSA by the mixed anhydride method (Erlanger et a/., 1957). After 48 hours of dialysis against distilled water at 4 C, the conjugate was further purified by 3 successive precipitations at iso-electric ph (- 4.8), centrifugations and redissolutions of the pellet in a 0.9 % saline solution, then freeze-dried. The ratio of steroid/mole of BSA was estimated from the difference in ultraviolet absorption as previously described (Erlanger et a/., The number of moles of hapten attached to each mole of protein was 13. c) Production of antiserum Antibodies were raised in 3 month old female New- Zealand white rabbits by injecting 1 mg of antigen dissolved in saline (ph 7) and emulsioned with 1 ml of complete Freund adjuvant, 4 times weekly, then 3 times every fortnight. Booster injections were thereafter given monthly. d) Estimation of the characteristics of the antibodies All experiments were made in a total volume of 400 wl of phosphate buffer (ph 7.4, 0.1 M, 0.1 % gelatine, 0.9 % sodium chloride, 0.1 % sodium azide) without or with increasing amounts of cold steroid, 100 ul of antiserum, 100 frl of radioactive steroid solution. Equilibrium was reached in 4 hours and remained inchanged for 24 hours. All experiments reported here have been performed with incubation times of hours at 4 C. Titers were measured as the reciprocal final dilution giving 50 % binding of the tracer. The mass of H-19 NT (19-(6,7(n)-!H) nor testosterone (19 Ci/mmol) (Amersham France SA Les Ulis Cedex) was equal to 135 pg. Specificity was investigated by measuring the relative cross-reactivity of 21 steroids or related compounds. The percent cross-reaction was calculated as the ratio of the mass of 19 NT (x 100) to the mass of the cross reacting compound which are required to displace 50 % of the labelled 19 NT. 2. Assay procedure a) Extraction and purification Plasma (1 ml) were extracted by ethyl-ether (10 ml). Further purification of the extracts was improved on Celite column chromatography (Chromatolithe A., Bio- Mérieux, Charbonni6res-les-Bains, France): 19 nor A4 androstenedione is eluated with 6 ml of isooctane, the column is then washed with again 6 ml of this solvant, then 19 NT is eluated with 6 ml of isooctane/ethyl-acetate (80/20). Purified extracts were dried and taken up to 1 ml with phosphate buffer. b) Radio-immunoassay of 19 NT 200 ul of sample extract, tritium labelled 19 NT and antiserum diluted at 1/10000 (200 fr1) were added successively. The standard curve was established using tubes containing 0, 25, 50, 100, 250, 500 and 1000 pg of 19 NT in 200 frl buffer. All the assays and standard were run in duplicate. After an overnight incubation at 4 C, 0.5 ml charcoal-dextran suspension (2.5 g charcoal and 0.25 g dextran per liter of buffer) was added at 4 C for 10 minutes and centrifuged at rpm for 15 min at 4 C. Five hundred microlitres of supernatant were mixed with 3 ml of scintillation cocktail and radioactivity was measured for 5 min. Non specific binding was determined in tubes that did not contain antibody. To avoid interassay variations, all plasma samples from a single animal were measured in the same assay. c) Radio-immunoassay of testosterone Testosterone was measured by a specific radioimmunoassay (reagents provided by Bio-M6rieux Charbonnieres-les-Bains, France). 3. Animals Blood samples from jugular and testicular veins were drawn simultaneously from adult (more than 4 years old) stallions during castration under general anesthesia. Each sample of blood was collected in heparinized vacutainer and rapidly centrifuged. Plasma was then removed and stored at - 30 C. Results Antisera of suitable titers were obtained from all animals within three months after beginning the immunization. The evolution of titers vs time was variable among the animals submitted to an identical immunization pattern (table 1). Because of the high titers of sera from the rabbit 1, these antibodies has been used for the development of radio-immunoassay of 19 NT.

4 Results of the study of cross-reaction of one antiserum (rabbit 1) are given in table 2. Among steroid tested only two had a significant crossreaction with 19 NT, namely 19 nor A4 androstenedione (90 %) and 19 nor testosterone acetate ( 100 %). Celite column chromatographie enable us to recover between 0 and 4 % of 19 nor A4 androstenedione in 19 NT fraction. The detection limit, calculated from the standard curve as the minimum amount of antigen that causes a significant decrease of bound radioactivity to antibodies, was 15 pg per tube which corresponds to about 150 pg/ml of plasma if taking extraction yield into account. Blank values determined using plasma extracts from castrated horses, were low enough to allow the detection of 1 50 pg/ml of plasma. Extraction yield was always controlled by radioactive tracer. Intra-assay and inter-assay coefficients of variation were 10% and 14 / respectively.

5 19 NT and testosterone concentration values obtained on horses plasmas are presented in table 3. Discussion The antibodies obtained by the method described above give only a low cross reaction with testosterone (0.1 %) and its major metabolites (0.1 to 0.5 %); whereas they show a complete cross reaction with 19 nor testosterone acetate and 19 nor A4 androstenedione. In fact, the linking of 19 NT with BSA takes place on C-17, this region being deeply entrenched in the albumin molecule cannot act as an antigenic determinant. A good specificity is therefore observed with regard to the estrene cycle, but not with regard to the nature or position of the substitutes in C-17. This absence of specificity is overcome by separation on a celite column, thus enabling the elimination of 19 nor A4 androstenedione. A binding of 19 NT to BSA by using a 3-0-carboxy-methyloxime (unpublished) enabled the author to obtain an excellent specificity with regard to the function carried on C-17 ; the antibodies thus produced present a relatively high cross-reaction with testosterone (6 %). As a preliminary separation of these two molecules is difficult, these antibodies have therefore not been selected for a radioimmunoassay of 19 NT. For methodological reasons during this experiment, it proved impossible to collect blood samples from the testicular artery, we will therefore assume that the hormonal concentration in the blood drawn from the jugular vein and the blood from the testicular artery are identical. The difference of 19 NT concentration observed in samples of jugular and testicular origin from horses A and B directly demonstrates the existence of a testicular secretion of 19 NT by the stallion. This result supports the findings of Samuels and Bedrack (1969) of a 19 NT biosynthesis from testosterone observed in the in vitro incubation of testicular homogenates from stallions. On the other hand this 19 NT secretion is not observed with animals C, D and E (although all animals were more than 4 year old); this absence is, however, correlated with a testicular secretion of testosterone much lower than those of horses A and B. As, the testicular hypoactivity during stress (particularly stress induced by anesthesia) in men (Me Intosh et al., 1981 ; Nakashima et al., 1975) and animals (Bambino and Hsueh, 1981 ; Kemppainen et al., 1983; Thibier and Rolland, 1976) has been previously described, it is logical assuming testicular localisation of 19 NT secretion to observe a depression of that secretion during such a surgical stress. Concentrations ratios for samples of testicular and jugular origin are quite higher for testosterone than 19 NT. It may result of a greater secretion rate of testosterone. In spite of these plasma concentrations levels are in the same order (about 500 and 800 pg/ml). It may be a consequence of a less important 19 NT metabolic clearance rate. Studies are now in progress to attempt to elucidate the mechanisms involved in these results. In conclusion, the results of this study confirm the existence of 19 NT in the stallion as demonstrated by Courtot et al. (1984) and support the secretion of this compound by the testis in that species. Received, 23rd January, Accepted, 28th March, Acknowledgements The 3H-19 nor testosterone used in this study was a gift of Laboratoires Theramex (France). This work was supported by a grant from «Service des Haras et de I Equitation». Summary Antiserum has been raised in rabbits treated with a 19 nor testosterone-hemisuccinate-bovine-serumalbumin conjugate and used for the development of a specific RIA of plasma 19 nor testosterone. Plasma samples are drawn from testicular and jugular veins of stallions during castration under general anesthesie. Results demonstrate a testicular secretion of 19 nor testosterone and a stress inhibition of this secretion correlatively with stress inhibition of testosterone secretion. References BAMBINO T.H., HSUEH A.J.W., Direct inhibitory effect of glucocorticoids upon testicular luteinizing hormone receptor and steroidogenesis in vivo and in vitro. Endocrinology, 108, COURTOT D., GUYOT J.L., BENOIT E., Mise en evidence de!elimination urinaire de la 19 nor-testosterone d origine endog6ne chez le cheval mate. C.R. Acad. Sci. Paris, 299,

6 ERLANGER B.F., BOREK F., BEISER S.M., LIEBERMAN S., Steroid-protein conjugates. 1. Preparation and characterization of conjugates of bovine serum albumin with testosterone and with cortisone. J. Bio/. Chem., 228, ERLANGER B.F., BOREK F., BEISER S.M., LIEBERMAN S., Steroid-protein conjugates. 2. Preparation and characterization of conjugates of bovine serum albumin with progesterone, deoxycorticosterone and estrone. J. Bio/. Chem., 234, HAMPL R., PICHA J., CHUNDELA B., Radioimmunoassay of nortestosterone and related steroids. J. Clin. Chem. Clin. Biochem., 17, JONDORF W.R., nortestosterone, a model for the use of anabolic steroid conjugates in raising antibodies for radioimmunoassay. Xenobiotica, 7, KEMPPAINEN R.J., THOMPSON F.N., LORENZ M.D., Effects of prednisone on thyroid and gonadal endocrine function in dog. J. Endocrinol., 96, Mc INTOSH T.K., LOTHROP D.A., LEE A., JACKSON B.T., NABSETH D., EGDAHL R.H., Circadian rhythm of cortisol is altered in postsurgical patients. J. Clin. Endocrinol. Metab., 53, NAKASHIMA A., KOSHIYAMA K., UOZUMI T., MONDEN Y., HAMANAKA K., Effects of general anaesthesia and severity of surgical stress on serum LH and testosterone in males. Acta Endocrinol., 78, THI81ER M., ROLLAND O., The effect of dexamethasone (DXM) on circulating testosterone (T) and luteinizing hormone (LH) in young postpubertal bulls. Theriogenology, 5,