Anti-Nandrolone monoclonal antibody, clone TUF2

The administration of steroids and other anabolic hormones to food producing animals for growth promotion purposes is strictly prohibited in the European Union. However, 17 alpha-19-nortestosterone (17 alpha-19-NT), the marker metabolite used for screening of the anabolic steroid 17 beta-19-nortestosterone (17 beta-19-NT, nandrolone), is recurrently detected in field urine samples from meat sheep by the Austrian Agency for Health and Food Safety, Competence Centre Veterinary Drugs and Hormones, Vienna, Austria. The aim of the present study was to obtain reference data for 17 beta-19-NT metabolites in plasma, urine, and feces systematically collected from pregnant ewes as well as from male and non pregnant female sheep before and after application of 17 beta-19-NT Samples were analysed by a regulatory HPLC-EIA and/or an LC-MS-technique. In plasma of pregnant ewes, neither 17 beta-19-NT nor corresponding metabolites could be detected. HPLC-EIA data showed an intermittent urinary excretion of 17 alpha-19-NT (up to 4.8 ng/ml) but not of 17 beta-19-NT In 4 out of 7 pregnant animals tested, maximum peaks of 17 alpha-19-NT were measured during the last 7 days before parturition (up to approx. 15 ng/ml). Also sporadic EIA-signals corresponding to 19-norandrostenedion e (19-NAD) were detected. Transiently increased signals corresponding to 17 alpha-19-NT and 19-NAD were also recorded in fecal samples from pregnant ewes throughout the gestation period (maximum concentrations 15 ng/g and 18 ng/g, respectively). In plasma of untreated young male and non pregnant female animals, concentrations of parent compound 17 beta-19-NT and related metabolites were constantly below the limit of detection (LOD). Analyses of urine revealed a sporadic excretion of 17 alpha-19-NT in untreated animals of either sex (up to 2.95 ng/ml) but not of 17 beta-19-NT and 19-NAD. Concentrations of both metabolites in selected feces samples were below the LOD. Following a single i.m. administration of nandrolone laurate (2 mg/kg b.m.), maximum mean concentrations of non conjugated 17 beta-19-NT were detectable in plasma on day 3 post injectionem (p.i.; 0.58 ng/ml in males, 0.44 ng/ml in females); only in single samples concentrations higher than the LOD were detectable up to 4 weeks p.i. Non conjugated metabolites were not detected. Urinary 17 beta-19-NT reached maximum mean concentrations of 17 ng/ml in males and 20 ng/ml in females at day 3 p.i. before the mean levels decreased to below the LOD by day 52 p.i. in females and by day 86 in males. For 17 alpha-19-NT maximum urinary concentrations were found at day 3 p.i. with mean values in males of 242 ng/ml and in females of 253 ng/ml. Concentrations continuously decreased thereafter and, by the last sampling date (day 226 p.i.), fluctuated at levels similar to those found before treatment. Episodic EIA-signals corresponding to 19-NAD standard were detected in urine for a period of 98 days in females and 114 days in males following 17 beta-19-NT application (with a maximum of 23 ng/ml). In fecal samples of treated animals, 17 alpha-19-NT and 19-NAD corresponding EIA-signals were only sporadically found at concentrations slightly higher than the LOD. The latter crossreacting analyte still awaits chemical identification. This study documents the natural occurrence of 17 alpha-19NT in urine from pregnant sheep and of young sheep of either sex. The 95- and 99-percentiles of the physiological 17 alpha-19-NT concentration were calculated to be 2.06 +/- 0.04 and 2.70 +/- 0.06 ng/ml, respectively. The detection of 17 alpha-19-NT in urine per se is, thus, not necessarily indicative of exogenous administration of 17 beta-19-NT. Using the established HPLC-EIA method feces turned out not to be a suitable matrix for 17 beta-19-NT surveillance purposes.

To exclude that aromatization plays a role in the estrogenic activity of tibolone, we studied the effect fibolone and metabolites on the aromatization of androstenedione and the aromatization of fibolone and its metabolites to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE) by human recombinant aromatase. Testosterone (T), 17alpha-methyltestosterone (MT), 19-nortestosterone (Nan), 7alpha-methyl-19-nortestosterone (MENT) and norethisterone (NET) were used as reference compounds. Sensitive in vitro bioassays with steroid receptors were used to monitor the generation of product and the reduction of substrate. LC-MSMS without derivatization was used for structural confirmation. A 10 times excess of tibolone and its metabolites did not inhibit the conversion of androstenedione to estrone by human recombinant aromatase as determined by estradiol receptor assay whereas T, MT, Nan, and MENT inhibited the conversion for 75, 53, 85 and 67%, respectively. Tibolone, 3alpha- and 3beta-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the Delta(4)-isomer suggests a conversion rate of 0.2% after 120 min incubation. In contrast T, MT, Nan, and MENT were completely converted to their A-ring aromates within 15 min while NET could not be aromatized. Aromatization of T, MT, Nan and MENT was confirmed with LC-MSMS. Structure/function analysis indicated that the 17alpha-ethynyl-group prevents aromatization of (19-nor) steroids while 7alpha-methyl substitution had no effect. Our results with the sensitive estradiol receptor assays show that in contrast to reference compounds fibolone and its metabolites are not aromatized. (C) 2002 Elsevier Science Inc. All rights reserved.