Evidence for the presence of endogenous 19-norandrosterone in human urine

Abstract

In 1997, in the scope of antidoping control in sport, a not inconsiderable number of urine analysed by official laboratories revealed the presence of 19-nortestosterone (19-NT: 17β-hydroxyestr-4-en-3-one) metabolites: 19-norandrosterone (19-NA: 3α-hydroxy-5α-estran-17-one) and 19-noretiocholanolone (19-NE: 3α-hydroxy-5β-estran-17-one). These repeated results on a short period of time generated some investigations and especially the verification of the possible production of these metabolites by an unknown endogenous route in adult entire male. Some experiences were led on different persons known to be non-treated with steroids and more precisely with nandrolone. Extractive methods were developed focusing on their selectivity, i.e. searching to eliminate at best matrix interferences from the target analytes. Gas chromatography coupled to mass spectrometry (quadrupole and magnetic instruments) was used to detect, identify and quantify the suspected signals. Two types of derivatization (TMS and TBDMS), a semi-preparative HPLC as well as co-chromatography proved unambiguously the presence, in more than 50% of the analysed urine (n=40), of 19-NA at concentrations between 0.05 and 0.60 ng/ml. 19-NE was not detected with the developed methods (LOD<0.02 ng/ml). Experiments led on athletes showed that after a prolonged intense effort, the 19-NA concentration can be increased by a factor varying between 2 and 4. Even if some complementary researches have to be done in order to determine the maximal physiological level of 19-NA and 19-NE, these results should considerably change the strategy of antidoping laboratories.

Introduction

The biological activity of 19-norandrostane derivatives was first studied by Dirscherl et al. in 1936 [1], [2] after synthesis of this class of compounds as a result of using the benzene ring and carbonyl group hydrogenation of estrone [3]. 19-Nortestosterone (17β-hydroxyestr-4-en-3-one) also named nandrolone was synthesised for the first time by Birch [4] in 1950 and by Wilds and Nelson [5], [6] in 1953. More recently, new synthetic procedures have been developed for the preparation of 19-norsteroids using, as starting material, compounds of the androstane serie [7], [8], [9]. The substitution of the C-19 methyl group by a hydrogen atom in testosterone changed appreciably the ratio between anabolic and androgenic activity. Certain 17-esters of nandrolone are much more clinically interesting than the compound itself, e.g. the phenylpropionate [10] or the decanoate [3]. This androgenic steroid is used in veterinary as well as in human medicine in cases of protein deficiency diseases, osteoporosis or burns. The use of this anabolic steroid as a doping agent to improve muscular strength and performance was rapidly extended in sport and horse racing because of its slight androgenic activity. 19-Nortestosterone has been also employed as a growth promoting agent to accelerate weight gain and improve feeding efficiency in animals [11], [12], [13].

Metabolism studies related to nandrolone or its esters have been realised in different species. Its metabolism in man was first investigated in 1958 by Engel et al. [14]. Two main metabolites were isolated after administering nandrolone to a postmenopausal breast cancer patient: 3α-hydroxy-5α-estran-17-one (19-norandrosterone) and 3α-hydroxy-5β-estran-17-one (19-noretiocholanolone) (Fig. 1). Although other metabolites are produced (19-norepiandrosterone), the control of their illegal administration is mainly based on the two first molecules [15], [16], [17], [18], [19]. According to Schänzer [19], metabolites of 19-nortestosterone in man are produced in the ratio 5α/5β of 72:28 (n=1), instead of 53:47 for testosterone. Houghton et al. studied the metabolism of 19-NT in horse and identified in urine the presence of glucuronic acid conjugate of 5α-estrane-3β,17α-diol, 5α-estrane-3β,17α-diol, 5α-estrane-3β,17β-diol and epinandrolone [20], [21]. In bovine, the metabolism studies showed that the most abundant metabolites were epinandrolone, 5α-estrane-3β,17α-diol and 5β-estran-3α-ol-17-one whatever the administration route [23], [24]. The most diagnostic analyte used to prove the illegal administration of 19-nortestosterone appears to be epinandrolone.

In the horseracing world, Courtot et al. [25] and Houghton et al. [26] found in 1984 that 19-NT was normally present in the urine of stallions; these preliminary results were confirmed later [27], [28]. Presence of nandrolone in equine follicular fluid was also demonstrated during the same period [29], [30]. The natural production of 19-NT and 19-norandrostenedione in the follicular fluid [29] and 19-NT in the plasma [31] of pregnant women was observed as well.

Belgian and Dutch researchers found that 17β-nandrolone was endogenously produced by intact boar [32], [33], [34], [35]; high concentrations (up to around 1 ppm) can be found in urine and minor levels were measured in edible parts. The first suspicion that 19-NT could also be endogenic in cattle was based on an artefact coming from 17α-testosterone (which was interpreted as 17α-nortestosterone because of poor methodology specificity) in the urine of male veal calves [37]. Meyer et al. explained in 1989 positive results in veal calve urine due to 19-nortestosterone in the feed, resulting from 19-NT contaminated bovine fat used for milk replacer production [38], [39]. Endogenous production hypothesis was made in 1991 by Vandenbroeck et al. in pregnant cow thanks to RIA analyses [36]. Meyer et al. identified in 1992 17α-nandrolone in the periparturient cow and the newborn calf urine [40]. Because of the important consequences for veterinary inspection in the EU, the EEC/DGVI asked officially to some official laboratories to confirm these results. The results obtained by GC–MS converged to the same conclusion: 17α-nortestosterone was unambiguously present in the cow peri-partum and the neo-natal calf urine [37], [41], [42]. Clouet et al. demonstrated the same phenomenon in pregnant sheep [43].

From all these discoveries, the new status of endogenous compound for nandrolone or its metabolites generated important consequences for the regulatory control. The strategy was systematically reviewed taking into account ratio between metabolites or concentrations. For instance, in horse, the international threshold for nandrolone in equine urine is reached when the ratio of 5α-estrane-3β,17α-diol to estr-5(10)-ene-3β,17α-diol exceeds one [22]. For meat inspection purposes, boars are no longer sampled for nortestosterone analysis; pig meat and derivative products are no longer controlled on 19-NT in trade between EU countries [37]. When epinandrolone is found in cow urine, a diagnostic of gestation is generally realised to exclude the possible endogenous origin.

Recently, a not inconsiderable number of nandrolone positive samples in adult males were found in a human anti-doping laboratory. The surprising results were not linked to the frequency of the positive cases but more to the sports which until that time had never been concerned by this scourge. Indeed, the involved disciplines were not until now linked to anabolic steroid consumption: judo, handball, football, and figure skating, etc. After having reproduced, with an independent method, the results of the first laboratory, the possible endogenous production of 19-NT main metabolites, i.e. 19-NA and 19-NE, was explored [44], [45]. For this purpose, a sensitive and specific GC–MS method was developed and validated on real samples. Healthy volunteer urine were then analysed at different moments of the day. Furthermore, the follow-up of individual male adults was realised before and after sport events. Finally, the profiles of more tendentious urine always subjected to some interrogations have been shown and discussed.