The use of steroids as doping substances has a long history. Especially anabolic steroids have been frequently used by professional athletes as well as in amateur sports. Most of the applied anabolic steroids have been derived from the male sexual hormone testosterone. Although the use of anabolic steroids is long known and a variety of different methods for proving the misuse of these substances are available in some cases it is still difficult to unequivocally determine whether a doping substance has been administered by an athlete or not. Sometimes the doping substance itself is no longer detectable within the body. Instead, some of the doping-metabolites can be detected for a limited period of time. Metabolites are formed in the human body by the action of different enzymes like cytochrome P450s or UDP glycosyltransferases (UGTs). In order to unequivocally convict athletes that have misused a doping substance by proving the presence of a doping-metabolite it is desirable to have a purified and structurally characterized sample of the doping-metabolite for reference purposes.
However, assessing some doping metabolites can be very difficult. For instance chemical synthesis is often cumbersome, time consuming or even fails due to the difficulties in generating metabolites containing e.g. regio-and stereospecific modifications.
In very close cooperation with the group of Prof. SchÃ¤nzer from the German Sport University in cologne our company, PomBioTech GmbH (www.pombiotech.com) have been able to successfully produce a metabolite of the anabolic steroid metandienone, named 17Î²-hydroxymethyl-17Î±-methyl-18-norandrosta-1,4,13-trien-3-one. This metabolite could not be synthesized via classical chemical synthesis. The scientific interest in this metabolite resulted in the fact that it is possible to detect it in the human body even after 19 days of its intake which is significantly longer compared to the detection period for other metandienone metabolites (usually up to 6 days). However, as mentioned before this metabolite could not be synthesized chemically disallowing its unequivocal structure elucidation. Moreover, no reference material was available making the proof for metandienone abuse by identifying this metabolite very difficult. The production of the metandienone metabolite was accomplished using a joint biotechnological and chemical synthesis approach. Since the in vivo generation of the metabolite in question probably involves the participation of several enzymes that catalyze different reactions, and a biotechnological rebuilding of this pathway was considered to be very demanding an artificial substrate that can be converted into the metabolite in a single enzymatic reaction was generated. This compound that is chemically derived from metandienone, 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one, was used as substrate for a biotechnological approach. The remaining enzymatic reaction was presumable catalyzed by a cytochrome P450 enzyme. Therefore, the biotechnological part of this approach involved the use of our patented cytochrome P450 expressing fission yeast cells. The recombinant fission yeast cells are cultivated together with the substrate, which is taken up by the cells, converted into the product of interest by the action of the expressed recombinant enzyme and is then secreted into media. By using our CYP21 expressing strain we were able to produce the metabolite of interest in sufficient quantities allowing a structure confirmation via NMR.
In conclusion, the combination of chemical and biotechnological methods has made it possible to produce and purify a doping metabolite that could previously not be synthesized via classical chemical approaches. Moreover, the joint chemical and biotechnological approach described above will lead to the production of other important doping metabolites in sufficiently high quantities allowing anti-doping laboratories worldwide to use these doping-metabolites as reference substances.
About Author / Additional Info:
Dr. Andy ZÃ¶llner, Researcher, Referent for biotechnology and head of marketing and IT at PomBioTech GmbH, has extensive experience with cytochrome P450 and other enzymes. For more information visit http://www.pombiotech.com/.