Reference ranges for urinary concentrations and ratios of endogenous steroids, which can be used as markers for steroid misuse, in a Caucasian population of athletes

Abstract

The detection of misuse with naturally occuring steroids is a great challenge for doping control laboratories. Intake of natural anabolic steroids alters the steroid profile. Thus, screening for exogenous use of these steroids can be established by monitoring a range of endogenous steroids, which constitute the steroid profile, and evaluate their concentrations and ratios against reference ranges. Elevated values of the steroid profile constitute an atypical finding after which a confirmatory IRMS procedure is needed to unequivocally establish the exogenous origin of a natural steroid. However, the large inter-individual differences in urinary steroid concentrations and the recent availability of a whole range of natural steroids (e.g. dehydroepiandrosterone and androstenedione) which each exert a different effect on the monitored parameters in doping control complicate the interpretation of the current steroid profile.

The screening of an extended steroid profile can provide additional parameters to support the atypical findings and can give specific information upon the steroids which have been administered.

The natural concentrations of 29 endogenous steroids and 11 ratios in a predominantly Caucasian population of athletes were determined. The upper reference values at 97.5%, 99% and 99.9% levels were assessed for male (n = 2027) and female (n = 1004) populations. Monitoring minor metabolites and evaluation of concentration ratios with respect to their natural abundances could improve the interpretation of the steroid profile in doping analysis.

Introduction

Testosterone (T), which was first purified and synthesised by Ruckzika and Butenandt in 1935 [1], [2] has played a major role in doping cases with anabolic steroids and in the development of new designer steroids. Based on the structure of T analogous compounds, both naturally occurring and newly developed synthetic steroids, were marketed and introduced in sports as anabolic agents. Starting from the 1970s with synthetic anabolic steroids and 1980s with T, the use of anabolic substances to enhance the physical capacities of athletes was prohibited by the International Olympic Committee (IOC) and International Sport Federations. The World Anti Doping Agency (WADA) also included the anabolic steroids in the prohibited list [3]. This ban led to extensive efforts to develop screening methods to detect misuse of anabolic steroids by anti-doping laboratories. Since the 1990s, the introduction of several endogenous steroids on the food supplement market, e.g. androstenedione (Adion), dehydroepiandrosterone (DHEA), androstanediol, provided easy access to prohormones via the internet. Besides these endogenous steroids, few exogenous designer steroids, e.g. tetrahydrogestrinone (THG), madol, were also marketed as food supplements.

In spite of the development of analytical tools to detect synthetic anabolic steroids in urine, the popularity of endogenous steroids as anabolic aid increased since the determination of the endogenous or exogenous origin from the substance is complicated. The latest WADA statistics of 2008 illustrate that this tendency still exists. Of more than 270,000 analysed doping samples world-wide last year, 5523 were returned as atypical finding (ATF) or adverse analytical finding (AAF) of which 2261 (40.9%) were caused by endogenous steroids.

For the detection of the administration of endogenous steroids, statistically based threshold values for selected screening parameters have been set [4], [5], [6], [7]. These parameters are primarily the parent steroids, which can be administered, such as T, epitestosterone (E), DHEA, Adion, dihydrotestosterone (DHT) and their most prominent metabolites like androsterone (Andro), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5ααβ-Adiol), 5β-androstane-3α,17β-diol (5βαβ-Adiol) [8], [9], [10]. Also the ratios of selected steroids, e.g. the testosterone to epitestosterone (T/E) ratio and the DHT to epitestosterone (DHT/E) ratio, are considered as valuable markers for the administration of endogenous steroids [5], [11], [12]. The combination of these individual steroid concentrations and ratios is traditionally referred to as the steroid profile. Currently it includes approximately 10 steroids which are generally quantified by GC/MS [13], [14]. When a screening procedure results in an atypical steroid profile, a GC/C/IRMS confirmation assay is applied which is based on isotopic differentiation between endogenous and exogenous steroids [15], [16], [17]. GC/C/IRMS cannot be applied to all samples due to technical limitations and hence the determination of the steroid profile still plays a crucial role in the detection of endogenous steroid misuse.

Because doping control laboratories receive anonymized samples, evaluation of an individual athlete's steroid profile must be based upon comparison with reference limits derived from population statistics. Since large inter-individual differences in urinary steroid concentrations exist, applications of small amounts of endogenous steroids might therefore remain unnoticed in a doping test.

Additionally, only a small number of studies have been published which include reference ranges for a limited number of steroids [6], [13], [18], [19], [20], [21]. These reference ranges are then applied in single steroid administration studies focussing on the urinary concentrations of a few steroid metabolites within a subject [11], [12], [22], [23].

Recently, minor metabolic pathways which lead to the formation of oxygenated and hydroxylated metabolites have been studied [10]. It has been shown that the application of endogenous steroids might saturate the main metabolic pathways thereby emphasising the formation of these minor metabolites [24]. This hypothesis underlines the importance of a comprehensive study of the minor metabolic pathways and the natural occurrence of these minor metabolites. The WADA technical document for endogenous steroids already includes some of these minor metabolites, e.g. 6α-OH-androstenedione (6α-OH-Adion), 6β-OH-androsterone (6β-OH-Andro), 6β-OH-etiocholanolone (6β-OH-Etio), 7β-OH-dehydroepiandrosterone (7β-OH-DHEA), 16α-OH-androsterone (16α-OH-Andro) and 7-keto-dehydroepiandrosterone (7-keto-DHEA) as examples of specific metabolites to detect the misuse of certain endogenous steroids [4]. Nevertheless the lack of reference ranges for these respective substances does not allow the analyst to unequivocally establish that a prohibited substance was taken [9]. Other minor metabolites like 4-OH-testosterone (4-OH-T), 4-OH-androstenedione (4-OH-Adion), 6α-OH-testosterone (6α-OH-T), 6α-OH-Adion, 16α-OH-androstenedione (16α-OH-Adion), 16α-OH-etiocholanolone (16α-OH-Etio) and 16α-OH-dehydroepiandrosterone (16α-OH-DHEA) have been identified in the course of several in vivo and in vitro application studies [25], [26], [27], [28], [29], [30], [31], [32]. Unfortunately, no data dealing with the normal urinary concentrations of these minor steroids in urine are available.

Therefore there is a compelling need to determine the reference ranges of a large number of endogenous steroids belonging to the ‘traditional’ steroid profile as well as to the new minor metabolic pathways based upon a large reference population in order to be helpful in the selection of urine samples for GC/C/IRMS analysis to prove misuse of natural steroids. Additionally, such a statistical study will provide more comprehensive insights into the steroid profile.