Plasma concentrations of testosterone and 19‐nortestosterone (nandrolone) in the nonracing intact male horse by liquid chromatography‐mass spectrometry

Abstract

The Commonwealth of Pennsylvania regulates the use of anabolic and androgenic steroids by monitoring plasma samples obtained from equine athletes post competition. Plasma samples were chosen over urine because the pharmacological action of any drug is generally based on plasma concentration of the parent or active metabolite of the compound and not its concentration in urine. Furthermore, the complex excretion pattern of anabolic and androgenic steroids makes urine a more difficult medium to work with. The androgens testosterone and nandrolone are endogenously produced in measurable concentrations in the intact male horse; therefore, the proposed regulation requires that a tolerance threshold be suggested for the intact male horse competing in an official race. Based on single IM administration, the plasma concentration of anabolic steroids was below the limit of quantification within 30 to 40 days (Soma et al., 2007), Anecdotal information suggests that these agents are typically administered every 2 to 3 weeks, it is suggested, until more information is available that veterinarians and trainers allow a minimum of 120 days withdrawal period following multiple administrations. Anabolic steroids are synthetic derivatives of the male hormone testosterone that have been modified to promote anabolic rather than androgenic actions. The anabolic effects are considered to be those promoting protein synthesis, muscle growth and erythropoiesis (Mottram & George, 2000). Anabolic steroids can exert strong effects on the body that may be beneficial for athletic performance (Hartgens & Kuipers, 2004). Androgenic steroids in the intact male horse include androstenedione, dihydrotestosterone, dehydroepiandrosterone, androstanediol, and testosterone, of which testosterone is the dominant steroid (Ganjam et al., 1973). The predominant method of quantification of androgenic steroids in plasma has been radioimmunoassay, which is not as specific as the direct measurement by liquid chromatography-mass spectrometry due to cross reactivity with other steroids (Silberzahn et al., 1988). Plasma concentrations of testosterone in mature normal males 27 months to 15 years of age measured by radioimmunoassay averaged 2000 pg ⁄mL (Inoue et al., 1993), concentrations ranging from 65 to 1600 pg ⁄mL have also been reported in the intact male and 15.3 ± 4.9 pg ⁄mL in geldings (Cox et al., 1973). Basal plasma testosterone concentrations showed seasonal variations with a low in January of 200 ± 100 pg ⁄mL to a high in April of 1400 ± 300 pg ⁄mL (Kirkpatrick et al., 1977; Aurich et al., 2003) and diurnal variation was also noted where concentrations were consistently lower at 18 h (Ganjam & Kenney, 1975). Using liquid chromatography-mass spectrometry for analysis, seasonal variation was also observed (Soma et al., 2007). Normal concentrations of testosterone and estrogen in intact male were attained by 16 months of age (Inoue et al., 1993) with plasma concentrations of testosterone increasing with age (Johnson et al., 1991). Stallions have androgen-to-estrogen conversion capabilities, therefore, intact male horses produce estrogens since testosterone is readily converted to estrogen by the horse testicle (Nyman et al., 1959). Intravenous administration of human chorionic gonadotrophin in horses with testicular tissue will stimulate a rise in testosterone (Cox et al., 1973) and estrogen (Zwain et al., 1989). In contrast, its injection into geldings will not produce the same effect, and castration will result in a rapid drop in both estrogen and testosterone (Ganjam & Kenney, 1975). Intramuscular administration of testosterone hexahydrobenzoate in the intact male produces a rapid rise in estrogen peaking in 24 h and a slower rise in testosterone with a peak concentration in 48 h (Zwain et al., 1989). Stallions can convert testosterone to estrogens (estrone and 17a-estradiol) by the Leydig cell of the testis that also produces Triple-stage quadrupole quantum mass spectrometer, Thermo Electron Corporation, San Jose, CA. J. vet. Pharmacol. Therap. 31, 587–590, doi: 10.1111/j.1365-2885.2008.00997.x. SHORT COMMUNICATION