CYP21-catalyzed production of the long-term urinary metandienone metabolite 17beta-hydroxymethyl-17 alpha-methyl-18-norandrosta-1,4,13-trien-3-one: a contribution to the fight against doping.
Anabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of metandienone, 17beta-hydroxymethyl-17 alpha-methyl-18-norandrosta-1,4,13-trien-3-one (20OH-NorMD), was discovered via LC-MS/MS and GC-MS. This metabolite was reported to be detected in urine samples up to 19 days after administration of metandienone. However, so far it was not possible to obtain purified reference material of this metabolite and to confirm its structure via NMR. Eleven recombinant strains of the fission yeast Schizosaccharomyces pombe that express different human hepatic or steroidogenic cytochrome P450 enzymes were screened for production of this metabolite in a whole-cell biotransformation reaction. 17,17-Dimethyl-18-norandrosta-1,4,13-trien-3-one, chemically derived from metandienone, was used as substrate for the bioconversion, because it could be converted to the final product in a single hydroxylation step. The obtained results demonstrate that CYP21 and to a lesser extent also CYP3A4 expressing strains can catalyze this steroid hydroxylation. Subsequent 5 l-scale fermentation resulted in the production and purification of 10 mg of metabolite and its unequivocal structure determination via NMR. The synthesis of this urinary metandienone metabolite via S. pombe-based whole-cell biotransformation now allows its use as a reference substance in doping control assays.
Zöllner
Andy
A
PomBioTech GmbH, D-66123 Saarbrücken, Germany.
Parr
Maria Kristina
MK
Drăgan
Călin-Aurel
CA
Dräs
Stefan
S
Schlörer
Nils
N
Peters
Frank T
FT
Maurer
Hans H
HH
Schänzer
Wilhelm
W
Bureik
Matthias
M
D045930:Anabolic Agents / Q000652:urine*; D001711:Biotransformation; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D004300:Doping in Sports / Q000517:prevention & control; D006801:Humans; D013058:Mass Spectrometry; D008696:Methandrostenolone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000652:urine*; D009654:Norsteroids / Q000378:metabolism* / Q000652:urine; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology; D013255:Steroid 21-Hydroxylase / Q000378:metabolism; D015813:Substance Abuse Detection / Q000379:methods
C547392:17-hydroxymethyl-17-methyl-18-norandrosta-1,4,13-trien-3-one; D045930:Anabolic Agents; D009654:Norsteroids; D011994:Recombinant Proteins; D008696:Methandrostenolone; D051544:Cytochrome P-450 CYP3A; D013255:Steroid 21-Hydroxylase; C510163:CYP3A4 protein, human