Urine Nandrolone Metabolites: False Positive Doping Test? R M N Kohler, M I Lambert
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325 REVIEW Urine nandrolone metabolites: false positive doping test? R M N Kohler, M I Lambert ............................................................................................................................. Br J Sports Med 2002;36:325–329 The aim of this review is to analyse the studies on Recently, the possibility of false positive tests for nandrolone metabolism to determine if it is possible for nandrolone has been raised. Explanations for false positive tests have included supplement an athlete to test positive for nandrolone without having contamination18 and endogenous production of ingested or injected nandrolone. nandrolone and regulation of metabolic pathways .......................................................................... of nandrolone metabolism by various physiologi- cal factors and supplement interventions. The aim of this review is to analyse the studies on he anabolic androgenic steroid 19- nandrolone metabolism with the overall goal of nortestosterone, also called nandrolone, was determining whether it is indeed possible for an Tfirst synthesised by Birch in 1950.1 Nan- athlete to test positive for nandrolone without drolone has an anabolic effect, and is used in the having either ingested or injected nandrolone. treatment of certain chronic diseases.1–3 The use of The question of a positive test resulting from nandrolone by athletes became popular in the late nutritional supplements14 18 and food 1950s.4 Athletes use nandrolone in an oral or contamination19 20 is beyond the scope of this injectable form to increase muscle strength and review. improve performance.5 As a result of the potential performance enhancing benefits6 and potential EVIDENCE FOR ENDOGENOUS NA health risks associated with anabolic steroid use,7 The origin of endogenous NA in the urine of ath- the International Olympic Committee (IOC) pro- letes who have not knowingly ingested or injected hibited the use of nandrolone in sport in 1976. nandrolone is central to resolving the question of whether it is possible to have a false positive test. The first study to suggest that NA could be found “A doping offence for nandrolone was in the urine of people free of exogenous defined as a concentration of NA in human nandrolone was a study on laboratory staff (n = urine exceeding 2 ng/ml in men and 5 14).8 Their urine was analysed using isotope dilu- ng/ml in women” tion mass spectrometry, and NA or a similar com- pound was suspected. This suspicion was based When nandrolone is ingested or injected by on the detection of a small peak for the ion at m/z 8 humans subjects, three metabolites of nan- 256. In retrospect, this signal may have been drolone can be isolated and measured in the urine caused by interference of other endogenous com- by gas chromatography-mass spectrometry. These pounds (noise) and perhaps represents a false metabolites have been identified as 19- positive finding. The authors acknowledged the norandrosterone (NA; 3α-hydroxy-5α-oestran- limitations of the study, the analytical technique 17-one), 19-noretiocholanolone (3α-hydroxy-5β- lacking specificity and sensitivity. oestran-17-one), and 19-norepiandrosterone Studies in 1988 and 1990 again raised the (3β-hydroxy-5α-oestran-17-one).8–13 These me- possibility of endogenous NA in the urine of 21 tabolites are isomeric compounds, having the humans. Kicman and Brooks used radioimmu- same chemical composition and molecular mass noassay and measured NA in the urine of men but different chemical structure. NA is usually the and women, who were supposedly free of most abundant urine metabolite of exogenous nandrolone, ranging from 3.8 to 49.4 nandrolone.9–13 The presence of these metabolites ng/ml. However, these data should be interpreted in the urine forms the basis of doping analysis for with caution as it could be argued that the the illegal use of nandrolone by athletes.8 12 14–16 analytical technique again lacks both specificity 11 This was based on the premise that these urine and sensitivity. Debruyckere et al measured NA metabolites could only have been derived from in the urine of three subjects at concentrations of 11 See end of article for exogenous nandrolone. A study in 1982 appeared 9, 14, and 37 ng/ml. These results were later authors’ affiliations to have found NA, or a similar compound, in the attributed to nandrolone contaminated meat 19 22 ....................... urine of athletes who had not used nandrolone.8 which the subjects may have eaten. Correspondence to: In 1996, the IOC stated that a critical concentra- In 1996, the IOC declared that the presence of a Dr Lambert, MRC/UCT tion for nandrolone metabolites in the urine had small amount of NA in the urine was not consid- 17 Research Unit for Exercise been established. A doping offence for nan- ered to constitute a doping offence. This Science and Sports suggests that they acknowledged the possibility Medicine, PO Box 115, drolone was defined as a concentration of NA in Newlands 7925, South human urine exceeding 2 ng/ml in men and 5 Africa; mlambert@ 17 ng/ml in women. ................................................. sports.uct.ac.za Abbreviations: IOC, International Olympic Committee; Accepted NA, 19-norandrosterone; GC-C-IRMS, gas 26 February 2002 “Recently, the possibility of false positive chromatography-combustion-isotope ratio mass ....................... tests for nandrolone has been raised.” spectrometry www.bjsportmed.com 326 Kohler, Lambert A recent study by Reznik et al25 examined the sequelae of giving human chorionic gonadotrophin to 10 men. Human chorionic gonadotrophin increases serum testosterone in healthy men41–43 and stimulates the aromatase enzyme, causing a gradual increase in serum oestrogen.41 44 45 The serum testosterone and oestrogen increased in the 10 subjects after human chorionic gonadotrophin administration, and NA excretion in the urine increased by 250%. It may be concluded from this study that the increase in nandrolone biosynthesis was possibly associated with the increased aromatisation of testosterone to oestrogen. Figure 1 The androgen biosynthetic pathway and the proposed aromatisation of androgen to oestrogen with the formation of 19-norsteroid intermediates. “Factors that could increase the flux of androgen precursors through the testosterone biosynthetic pathway could theoretically increase the amount of of endogenous NA production. It can only be assumed that nandrolone produced.” this decision was reached on the basis of data collected by IOC laboratories during routine drug testing, as the scientific evi- Although the pathways proposed are theoretical, the avail- dence at the time was equivocal. In the late 1990s, analytical able evidence suggests that it is possible for the flux of andro- procedures for the detection and quantification of steroid 23 gen precursors through the testosterone biosynthetic pathway metabolites in urine had become increasingly sensitive. This to result in the production of endogenous nandrolone. There- may have accounted for an appreciable number of positive fore it can be assumed that factors that could increase the flux urine samples for NA being analysed in certain anti-doping of androgen precursors through the testosterone biosynthetic laboratories. Many of the positive samples were from partici- pathway could theoretically increase the amount of nan- pants of sports that had previously not been associated with drolone produced. anabolic steroid use.1 Further research with more sensitive equipment was undertaken to determine if NA could be pro- duced naturally by the human body. This research showed FACTORS WITH THE POTENTIAL TO AFFECT NA convincing evidence that NA was found in the urine of METABOLISM subjects free of exogenous nandrolone.1 15 18 24–28 The urine NA Genetics concentrations in these studies ranged from 0.01 to 1.79 There is a wide range of serum testosterone concentrations in ng/ml. In a study by Galan Martin et al,29 high NA concentra- men,32 suggesting large genetic interindividual and intraindi- tions in five sportspeople (4, 5, 6, 8, and 14 ng/ml) were vidual variability in sex steroid production and excretion over measured. One woman in the study, who was postmeno- a 24 hour period.46 The possibility therefore exists that there is pausal, had a NA concentration of 22 ng/ml. It could be argued a variable rate of NA excretion.1 Indeed, endogenous NA urine that these athletes had administered nandrolone.29 These excretion in a male athlete varied by 680% over a three month results are difficult to explain and perhaps further investiga- period1 and in another subject by 72% over a 24 hour period.15 tion of these subjects is necessary before a definite opinion can The enzyme complex 17β-hydroxysteroid dehydrogenase, be formed. which is responsible for converting androstenedione into tes- tosterone, and the aromatase enzyme complex, which converts testosterone into oestrogen, occur in muscle and METABOLISM OF NA fat.31 47 Therefore, it is conceivable that people with higher Aromatisation muscle and fat content may be more proficient in the produc- Metabolic pathways for the endogenous production of NA in tion of 19-norsteroid intermediates. The aromatase enzyme the human body need to be considered. Under normal complex per se can also show considerable genetic variability circumstances, testosterone is aromatised to oestrogen by the in expression and activity in certain people,48 with increased aromatase enzyme complex.30 Androstenedione, the direct activity of the aromatase enzyme producing larger amounts