CYP21-catalyzed production of the long-term urinary metandienone metabolite 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one: A contribution to the fight against doping

Article abstract of DOI:10.1515/BC.2010.002

Anabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of metandienone, 17β-hydroxymethyl-17α-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. by Walter de Gruyter.

Full text of DOI:10.1515/BC.2010.002

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Biol. Chem., Vol. 391, pp. 119–127, January 2010 • Copyright ꢀ by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2010.002
CYP21-catalyzed production of the long-term urinary
metandienone metabolite 17b-hydroxymethyl-17a-methyl-18-
norandrosta-1,4,13-trien-3-one: a contribution to the fight
against doping
Andy Zo¨llner1,a, Maria Kristina Parr2,a,*,
Keywords: anabolic steroids; CYP21; CYP3A4; doping;
metabolite; metandienone; Schizosaccharomyces pombe;
whole-cell biotransformation.
Caˇlin-Aurel Draˇgan1, Stefan Dra¨s1, Nils Schlo¨rer3,
Frank T. Peters4,5, Hans H. Maurer4, Wilhelm
Scha¨nzer2 and Matthias Bureik1
1 PomBioTech GmbH, D-66123 Saarbru¨cken, Germany
Introduction
2 Institute of Biochemistry, German Sport University,
D-50933 Cologne, Germany
Cytochromes P450 (P450s or CYPs) form a remarkably ver-
satile enzyme superfamily and are involved in a variety of
different reactions. These hemoproteins are spread through-
out plants, animals and microorganisms where they partici-
pate in crucial physiological processes such as the
biodegradation of xenobiotics during phase-I metabolism,
steroidogenesis, bile acid synthesis or the metabolism (and
sometimes even the activation) of a variety of carcinogenic
substances. The reactions catalyzed by these enzymes
include hydroxylation, N-, O- and S-dealkylation, sulfoxi-
dation, epoxidation, deamination and N-oxide reduction. In
view of their high substrate diversity it is not surprising that
P450s have attracted the attention of different research fields
such as biochemistry, pharmacology, physiology, organic
chemistry and biotechnology (Bernhardt, 2005, 2006; Isin
and Guengerich, 2007).
The human CYP superfamily comprises 57 members that
are divided into 18 families and 43 subfamilies; among these,
members of families 1–3 mediate 70–80% of all phase I-
dependent metabolism of clinically used drugs and partici-
pate in the metabolism of a huge number of other xenobiotic
chemicals (Guengerich, 2005; Ingelman-Sundberg et al.,
2007). Depending on the subcellular localization of the
respective P450 system, the electrons required for the activity
of human P450s are provided in two different ways (Han-
nemann et al., 2007): mitochondrial P450s (which are mainly
involved in steroidogenesis) obtain the two electrons needed
for a hydroxylation reaction one by one via a short electron
transfer chain consisting of the w2Fe-2Sx-protein adrenodoxin
and the FAD-containing NADPH-dependent adrenodoxin
reductase, whereas microsomal CYPs (which play an impor-
tant role in the degradation of a variety of different drugs,
alkaloids and pesticides in the human liver) receive both
electrons directly from the FAD- and FMN-containing cyto-
chrome P450 reductase (CPR).
3 Institute of Organic Chemistry, University of Cologne,
D-50939 Cologne, Germany
4 Department of Experimental and Clinical Toxicology,
Saarland University, D-66421 Homburg/Saar, Germany
5 Institute of Forensic Medicine, Friedrich Schiller
University, D-07740 Jena, Germany
*Corresponding author
e-mail: m.parr@biochem.dshs-koeln.de
Abstract
Anabolic-androgenic steroids are some of the most frequent-
ly misused drugs in human sports. Recently, a previously
unknown urinary metabolite of metandienone, 17b-hydroxy-
methyl-17a-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 Schizosac-
charomyces pombe that express different human hepatic or
steroidogenic cytochrome P450 enzymes were screened for
production of this metabolite in a whole-cell biotransforma-
tion 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 convert-
ed 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 ster-
oid 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.
Owing to their significant contribution to the metabolism
of anabolic steroids, P450s have also been the focus of anti-
doping laboratories. Previous investigations have shown that
a variety of different P450s (including CYP3A4, the pre-
dominant liver P450) are capable of hydroxylating anabolic
a These authors contributed equally to this work.
2010/257
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120 A. Zo¨ llner et al.
Therefore, a set of S. pombe strains that express different
human hepatic or steroidogenic P450s was used to screen for
the formation of 20OH-NorMD. Using P450 expressing fis-
sion yeast strains for screening purposes instead of micro-
some preparations enables a direct production of the
metabolite of interest in the milligram to gram range and
facilitates the establishment of an upscaling process.
Figure 1 Reaction scheme for the CYP-dependent formation of
20OH-NorMD from 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-
one.
Results
steroids at various positions. For example, CYP3A4 is main-
ly responsible for the 6b-hydroxylation of testosterone and
of synthetic anabolic steroids such as metandienone (Scha¨n-
zer, 1996; Rendic et al., 1999; Walsky and Obach, 2004).
The latter steroid has been the focus of attention of anti-
doping researchers in the past number of years because sev-
eral professional athletes were convicted of misusing this
substance for doping purposes. Commonly used methods to
detect the abuse of this anabolic steroid are based on the
identification of different metandienone metabolites via LC-
MS/MS and GC-MS analysis (Scha¨nzer et al., 1991, 2006;
Scha¨nzer and Donike, 1993; Scha¨nzer, 1996; Rendic et al.,
1999). Among these metabolites is a recently identified urinary
metabolite, 17b-hydroxymethyl-17a-methyl-18-norandrosta-
1,4,13-trien-3-one (also referred to as ‘20OH-NorMD’), that
allows the long-term detection of metandienone abuse up to
19 days after a single administration of 5 mg of the drug.
This time span is significantly increased compared to the
detection period of other metandienone metabolites reported
detectable only between 4 and 6 days (Scha¨nzer et al., 2006).
Although the identification of 20OH-NorMD in urine sam-
ples by comparison with post-administration urines from
controlled metandienone excretion studies is accepted by the
WADA (World Anti-Doping Agency) as proof of metandie-
none abuse, it has not yet been possible to finally confirm
its structure. Moreover, this metabolite was not available as
purified reference substance because its chemical synthesis
was not achieved. Therefore, it was the aim of this study to
obtain 20OH-NorMD in sufficiently high quantities to allow
both NMR structure identification and its use as reference
material.
The in vivo formation of 20OH-NorMD from metandie-
none probably involves the participation of several enzymes
that catalyze different reactions, and a biotechnological
rebuilding of this pathway was considered to be very
demanding. Therefore, 17,17-dimethyl-18-norandrosta-1,4,
13-trien-3-one, a compound that is chemically derived from
metandienone, was used as substrate because it can be con-
veniently synthesized and requires only a single hydroxyla-
tion reaction for its conversion into 20OH-NorMD (Figure
1). Moreover, it seems plausible to assume that this final
reaction step is catalyzed by a P450 enzyme. Recombinant
strains of the fission yeast Schizosaccharomyces pombe have
been successfully used for the expression of a variety of
different human CYPs as well as for the biotechnological
production of P450 metabolites in whole-cell biotransfor-
mation assays (Bureik et al., 2002; Dragan et al., 2005,
2006b; Peters et al., 2007, 2009a,b; Zo¨llner et al., 2009).
Cytochrome P450 screening assays
Eleven fission yeast strains that recombinantly express hepat-
ic or steroidogenic CYPs (CYP2B6, CYP2C9, CYP2C19,
CYP2D6, CYP3A4, CYP3A7*2, CYP4Z1, CYP11B1,
CYP11B2, CYP17 and CYP21, respectively) were tested for
the bioconversion of chemically synthesized 17,17-dimethyl-
18-norandrosta-1,4,13-trien-3-one to 20OH-NorMD (Figure
1). Recent studies indicate that coexpression of human CPR
enhances the biotransformation activity of fission yeast
strains that express human microsomal P450s compared with
strains using only the endogenous CPR of S. pombe (Dragan
et al., 2006b; Peters et al., 2007, 2009a,b). For the CYP17-
and CYP21-dependent biotransformations we therefore used
the CPR coexpressing strains CAD71 and CAD75 (Dragan
et al., manuscript in preparation) instead of the previously
described strains CAD8 and CAD18 (Dragan et al., 2006a,b)
that solely express human CYP17 or CYP21, respectively.
In addition to strains expressing the major liver P450s we
also tested strain INA1 (Neunzig et al., submitted) which
expresses CYP3A7*2, a polymorphic variant of the fetal
enzyme CYP3A7 that displays improved activity towards
some substrates (Rodriguez-Antona et al., 2005).
The performed screening assay showed that the CYP21
expressing S. pombe strain CAD75 was the most efficient
one to catalyze the desired hydroxylation reaction (Table 1).
The EI mass spectrum of trimethylsilylated (TMS) product
of this biotransformation displayed the typical ion fragmen-
tation pattern of 20OH-NorMD (Scha¨nzer et al., 2006). In
comparison to 20OH-NorMD (Mqs442) the analyzed sub-
strate shows Mqs354. In addition, another hydroxylated
metabolite was also produced by CYP21 in equal or even
higher quantities than 20OH-NorMD. It also shows Mqs442
but in contrast to 20OH-NorMD no loss of 103 u (CH2-O-
TMS) is observed. The mass spectra of the TMS derivatives
of the above-mentioned steroids are displayed in Figure 2.
CYP3A4, which is reported to be the most abundant P450
in human liver, was also capable of producing the desired
metabolite together with its 17-epimer 17a-hydroxymethyl-
17b-methyl-18-norandrosta-1,4,13-triene-3-one (Table 1),
which has been previously identified in urine samples
(Scha¨nzer et al., 2006; Parr et al., 2007). Moreover,
CYP2C19 also metabolizes the substrate. However, the
formed metabolite was not 20OH-NorMD and the amount
of this CYP2C19 synthesized compound was too low to
allow any further investigations. With all other tested P450
expressing fission yeast strains (i.e., CYP2B6, CYP2C9,
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CYP21 catalyzed metandienone metabolite production 121
Figure 2 EI mass spectra of (A) trimethylsilylated 17b-hydroxymethyl-17a-methyl-18-norandrosta-1,4,13-trien-3-one, (B) trimethylsily-
lated 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one and (C) 16b-hydroxy-17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one.
CYP2D6, CYP3A7*2, CYP4Z1, CYP11B1, CYP11B2 and
CYP17, respectively) no products of 17,17-dimethyl-18-
norandrosta-1,4,13-trien-3-one were observed.
1 g/l during the initial growth phase of the cells (40 h) and
the relative oxygen content within the fermentation broth
steadily decreased from initially 100% to 20%. Preliminary
small-scale experiments indicated that substrate concentra-
tions between 175 and 225 m
M lead to the highest product
Large scale whole-cell biotransformation
formation ratio, whereas higher amounts of substrate inhibit
product formation (data not shown), an observation that has
also been made with other P450s (Korzekwa et al., 1998;
Lin et al., 2001). For the biotransformation the substrate was
therefore added in two batches of 150 mg each (at an interval
For the production of larger amounts of 20OH-NorMD, a
5 l-scale fed-batch fermentation was performed using the
CYP21 expressing strain CAD 75 (Figure 3). In this process,
the glucose concentration was maintained at approximately
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122 A. Zo¨ llner et al.
Table 1 Results of the cytochrome P450 screening assays using recombinant S. pombe strains.
Cytochrome
P450
Positive control reaction
Observed 17,17-dimethyl-18-
norandrosta-1,4,13-
expressed in
S. pombe
trien-3-one metabolites
CYP2B6
CYP2C9
CYP2C19
CYP2D6
CYP3A4
Bupropion™hydroxybupropion
n.d.
n.d.
Tolbutamide™49-hydroxymethyltolbutamide
S-Mephenytoin™49-hydroxy-S-mephenytoin
Dextromethorphan™dextrorphan
Unidentified metabolite
n.d.
Testosterone™6b-hydroxytestosterone
(A) 20OH-NorMD
(B) 17a-Hydroxymethyl-17b-
methyl-18-norandrosta-
1,4,13-triene-3-one
n.d.
n.d.
n.d.
n.d.
n.d.
(A) 20OH-NorMD
(B) 16b-Hydroxy-17,17-
dimethyl-18-norandrosta-
1,4,13-trien-3-one
CYP3A7*2
CYP4Z1
CYP11B2
CYP11B1
CYP17
DHEA™16a-hydroxy-DHEA
Myristic acid™hydroxymyristic acids
Deoxycorticosterone™corticosterone
Deoxycortisol™cortisol
Progesterone™17a-hydroxyprogesterone
17a-Hydroxyprogesterone™deoxycortisol
CYP21
Only the CYP2C19, CYP3A4 and CYP21 expressing fission yeast strains were capable of metab-
olizing the substrate 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one. 20OH-NorMD was most
efficiently produced by the human CYP21 expressing strain. All monitored standard reactions are
indicated.
n.d.sno detectable product formation.
of 24 h) and the glucose concentration was increased, where-
because an exact quantification of the product was not pos-
sible owing to the lack of reference material. Analysis of the
relative peak areas of substrate and both major metabolites
formed by the CYP21 expressing strain at the fermentation
endpoint indicates that approximately 10% of the substrate
was converted to the desired metabolite and 30% to the
byproduct.
as the relative oxygen content remained approximately con-
stant at around 20%. Because P450-catalyzed reactions
require NADPH, which is predominantly formed in the pen-
tose phosphate pathway, it is necessary that glucose is present
in sufficiently high amounts in the fermentation culture. As
shown in Figure 3, the biomass dry weight at the fermenta-
tion end was approximately 10 g/l. The product formation is
expressed as relative areas of product obtained from total
ion chromatograms after GC-MS analysis and was monitored
at different time points as indicated in Figure 3. This approx-
imate estimation of the product formation rate was necessary
Product purification and NMR analysis
After extraction of the steroids from the fermentation broth
using ethyl acetate and removal of remaining substrate, the
Figure 3 Whole-cell biotransformation of the CYP21 expressing S. pombe strain CAD75 in a 5 l stirred-tank fermenter.
Fermenter volume (dashed line), dissolved oxygen concentration (solid gray line), glucose concentration (dotted line with open triangles),
biomass dry weight (dw; solid black line with open circles) and product formation (solid black line with filled boxes) were monitored
throughout the fermentation process. Relative product peak areas were determined from total ion chromatograms obtained after GC-MS
analysis and normalization against the internal standard metandienone.
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CYP21 catalyzed metandienone metabolite production 123
without purified reference material has been accepted by the
WADA as proof for metandienone abuse, until now, the ath-
letes in question could challenge the results of the anti-dop-
ing laboratories resulting in prolonged trials. The results
presented in this study demonstrate a successful combination
of chemical methods (substrate synthesis) and biotechnolog-
ical tools (whole-cell biotransformation) that allowed the
synthesis of 20OH-NorMD which could not be accomplished
by other means. The availability of 20OH-NorMD as refer-
ence compound will provide an incontestable proof for
metandienone abuse even after 19 days of its intake helping
anti-doping laboratories to identify athletes who have con-
sumed the anabolic steroid metandienone, particularly during
training periods. Because metandienone is usually not
administrated during official competitions but during training
periods the facilitated and unequivocal long-term detection
of metandienone abuse will help to convict athletes who for-
merly had escaped conviction owing to the shorter detection
periods of other metandienone metabolites. Thus, the avail-
ability of 20OH-NorMD as reference material is of great
significance in the fight against doping.
As mentioned above, human liver CYPs clearly play an
important role in the metabolism of anabolic steroids and,
consequently, in the detection of steroid abuse (Scha¨nzer,
1996; Rendic et al., 1999). But the activity of steroidogenic
CYPs also needs to be considered, as is exemplified by the
involvement of CYP19 in nandrolone biosynthesis (Reznik
et al., 2001) and the influence of CYP17 polymorphisms on
the testosterone/epitestosterone glucuronide ratio (Schulze
et al., 2008). The results of the present study suggest that the
CYP21-dependent metabolization of the metandienone
metabolite 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one
is added to this list. From an enzymatic point of view, it is
very interesting to identify both a new substrate and new
stereo-selective properties of human CYP21. To the best
of our knowledge, the only activities that have so far been
demonstrated for this enzyme are the 21-hydroxylations of
progesterone and 17a-hydroxyprogesterone (Lorence et al.,
1989; Wu et al., 1991). The CYP21-dependent steroid b-
hydroxylations observed in this study indicate that the activ-
ity of this enzyme (like its closest human homolog CYP17)
is not as strictly stereo- and regio-specific as has been
thought previously. Thus, we might see other CYP21 metab-
olites of both natural and artificial steroids in the future.
The findings presented in this study naturally raise the
question of whether CYP21 and CYP3A4 are actually both
involved in the in vivo formation of 20OH-NorMD or not
and, if so, to what extent each of them contributes to the
production of this urinary metandienone metabolite over
time. In this context, it is helpful to emphasize the fact that
each of the two CYPs produces a second metabolite from
17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one that is not
formed by the other enzyme. Thus, the involvement of
CYP3A4 seems to be very likely because the epimeric form
of 20OH-NorMD (which was not observed in the CYP21-
dependent biotransformations) has been previously identified
in urine samples (Scha¨nzer et al., 2006; Parr et al., 2007). In
Figure 4 Chromatogram of the semi-preparative HPLC purifica-
tion of 20OH-NorMD.
20OH-NorMD (solid black arrow) as well as 16b-hydroxy-17,17-
dimethyl-18-norandrosta-1,4,13-trien-3-one (dashed arrow) are sep-
arated from other byproducts and compounds generated by the
CYP21 expressing S. pombe strain CAD75. Substrate remainder was
previously removed as described in the materials and methods sec-
tion. The product was isocratically eluted from the semi-preparative
column wNucleosil N(CH3)2, Macherey-Nagelx using n-hexane:2-
propanol (95:5) as solvent at 5 ml/min.
final purification step was performed by semi-preparative
HPLC. To identify the optimal purification conditions a vari-
ety of different HPLC materials and eluent compositions
were tested wRP-C18, RP-C8 and N(CH3)2 column materials
from Macherey and Nagel, Du¨ren, Germanyx. The best
results were obtained using a N(CH3)2 column with n-hex-
ane:isopropanol (95:5 v/v) as solvent. Under isocratic con-
ditions the product was eluted after approximately 450 s,
whereas the byproduct was eluted from the column after
approximately 420 s (Figure 4). In this way, 10 mg of the
product as well as 10 mg of the byproduct were isolated.
1
Subsequent NMR analysis w H NMR (600 MHz, CDCl3)
d: 2.02/1.59 (m, 2H, H-16), 1.24 (s, 3H, H-19), 0.95 (s, 3H,
H-20a), 3.51/3.37 (d, 2H, H-20b); 13C NMR (150 MHz,
CDCl3) d: 137.5 (C-13), 138.9 (C-14), 33.8 (C-16), 51.6
(C-17), 18.5 (C-19), 21.7 (C-20a), 68.9 (C-20b)x unequi-
vocally confirmed the proposed structure of 20OH-NorMD
as shown in Figure 1. Moreover, NMR analysis of the bypro-
duct revealed that this CYP21 metabolite is b-hydroxylated
1
at position C-16 w H NMR (600 MHz, CDCl3) d: 3.94 (dd,
1H, H-16), 1.16 (s, 3H, H-19), 0.99 (s, 3H, H-20a), 0.91 (s,
3H, H-20b); 13C NMR (150 MHz, CDCl3) d: 140.7 (C-13),
131.5 (C-14), 80.3 (C-16), 47.9 (C-17), 18.5 (C-19), 24.6
(C-20a), 18.8 (C-20b)x.
Discussion
Although the identification of the long-term urinary metan-
dienone metabolite 20OH-NorMD in doping control samples
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124 A. Zo¨ llner et al.
contrast, the occurrence of the CYP21-specific 16b-hydroxy
metabolite in urine samples of metandienone abusers still
needs to be investigated. But even the participation of other
human enzymes (CYPs or others) that were not included in
this study cannot be ruled out. However, assessing these
questions was not within the scope of the present study.
Other possible approaches to clarify the physiological role
of CYP21 and CYP3A4 in the formation of 20OH-NorMD
could either involve administration studies, e.g., including
voluntary patients exhibiting a mild or non-classic CYP21
deficiency (congenital adrenal hyperplasia, CAH) (White and
Speiser, 2000), the use of adequate animal models, the inves-
tigation of primary hepatic as well as adrenal cell cultures,
or the examination of microsome preparations of the respec-
tive tissues.
The clinical manifestations of classic CAH are very severe
ranging from patients suffering from extreme salt wasting,
owing to the incapability of producing cortisol and aldoster-
one, the most important gluco- and mineralocorticoids, to a
virilization of females caused by the excessive production of
androgens (White and Speiser, 2000). The symptoms of the
mildest from of CAH, non-classic CAH, are premature
pubarche in children (Temeck et al., 1987), severe cystic acne
(Marynick et al., 1983) and oligomenorrhea in young women
(Rosenwaks et al., 1979). Only patients with a mild or non-
classic CAH can be ethically suited for a metandienone
administration study. However, owing to the mild clinical
symptoms, patients with non-classical 21-hydroxylase defi-
ciency very often remain undetected (White and Speiser,
2000).
cell lines could be used to examine the role of CYP3A4
(Martinez-Jimenez et al., 2005). In these cell culture exper-
iments, the use of known CYP3A4 inducers and inhibitors
could help to elucidate the role of CYP3A4 in the formation
of 20OH-NorMD.
If both CYP21 and CYP3A4 are found to be relevant for
in vivo formation of 20OH-NorMD, it will be interesting to
investigate to what extent each of them contributes to this
reaction during the time span when the metabolite is detect-
able. As each of the fission yeast strains CAD67 and CAD75
strongly expresses the respective CYP gene under control of
the strong endogenous nmt1 promoter, the biotransformation
rates observed in this study do not reflect the situation in the
human body where the expression of the two genes is inde-
pendently controlled by their native promoters in different
tissues. Thus, in man the contribution of CYP3A4 to 20OH-
NorMD formation might even be higher than that of CYP21.
In conclusion, by combining chemical and biotechnolog-
ical methods it has been possible to produce and purify the
long-term metandienone metabolite 20OH-NorMD in suffi-
cient amounts to allow its structure confirmation by NMR.
Two human P450s, CYP3A4 and CYP21, were identified as
likely candidates for catalyzing the in vivo formation of this
compound. Moreover, the techniques described in this paper
will lead to the production of this important metabolite in
sufficiently high quantities to allow anti-doping laboratories
worldwide to use this urinary metabolite as reference
substance.
Another possible approach to confirm the physiological
participation of CYP21 in the formation of 20OH-NorMD
might be the use of a mouse model. A naturally occurring
mouse strain with a CYP21 deletion has been previously
reported (Tajima et al., 1999), and a comparison of metan-
dienone metabolization results obtained from this strain with
those of wild type mice could help to clarify a possible role
of CYP21. The use of primary human adrenal cells of
healthy and CAH patients could also be used to assess this
question. However, owing to their low proliferation rate pri-
mary human adrenocortical cells are difficult to obtain and
to cultivate (Cardoso et al., 2009). Moreover, because extra-
renal CYP21 activity has been reported before, e.g., in lym-
phocytes and heart tissue (Zhou et al., 1997; Kayes-
Wandover and White, 2000; Forest, 2004), such experiments
might not suffice to unequivocally clarify the role of CYP21
in the formation of 20OH-NorMD.
An investigation of the role of CYP3A4 could be con-
ducted in similar ways as proposed for CYP21. For instance,
administration studies on volunteers exhibiting a decreased
CYP3A4 activity caused for example by a gene polymor-
phism (Pirmohamed and Park, 2003) or mutated CYP3A4
promoter regions (Ingelman-Sundberg et al., 2007) are an
option. Moreover, the Cyp3A-knockout mouse model gen-
erated by van Herwaarden that lacks all 13 mouse CYP3A
genes (van Herwaarden et al., 2007) seems to be a suited
tool for such an investigation. In addition, primary HepG2
Materials and methods
Chemicals
17a-Hydroxymethyl-17b-methyl-18-norandrosta-1,4,13-trien-3-one
was synthesized as previously described (Parr et al., 2007). 17,17-
Dimethyl-18-norandrosta-1,4,13-trien-3-one was chemically derived
as follows: 1 g of metandienone was dissolved in 40 ml of a mixture
of methanol: 1 N hydrochloric acid (1:1) and refluxed for 16 h. The
product was extracted three times with 50 ml of n-pentane. All other
steroids and reference substances used in this study were purchased
from Sigma (Deisenhofen, Germany).
General techniques, media and strains
General DNA manipulation methods were performed according to
standard techniques (Sambrook et al., 1989). Media and genetic
methods for studying fission yeast were performed according to
standard procedures (Moreno et al., 1991; Alfa et al., 1993). Unless
indicated otherwise, strains were cultivated at 308C in Edinburgh
minimal medium (EMM) with 20 g/l glucose and supplements as
required. The fermentation medium DM1 (1 l) was composed of
2.2 g Na2HPO4, 5 g NH4Cl, 6 g KH2PO4, 22 ml salt stock solution
(Moreno et al., 1991), 1 ml vitamin stock solution (Moreno et al.,
1991) and 100 ml mineral stock solution (Moreno et al., 1991). In
all constructs, expression of the human CYPs is regulated by the
thiamine-repressible nmt1 promoter (Maundrell, 1990) of fission
yeast. Thiamine was used at a concentration of 5 m
All strains are listed in Table 2.
M throughout.
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CYP21 catalyzed metandienone metabolite production 125
Table 2 Recombinant fission yeast strains expressing human cytochrome P450 systems.
Strain
Genotype
Expressed
protein(s)
Standard
substrate(s)
References
NCYC2036
CAD62
AZ3
h- ura4.dl18
–
CPR
CPR,
CYP4Z1
CPR,
CYP2D6
CPR,
CYP2B6
CPR,
CYP2C19
CPR,
CYP3A4
CPR,
CYP2C9
CPR,
CYP17
CPR,
CYP21
CPR,
CYP3A7*2
CYP11B2
–
–
Losson and Lacroute, 1983
Dragan et al., submitted
Zo¨llner et al., 2009
h- ura4.dl18::pCAD1-CPR
h- ura4.dl18::pCAD1-
CPR/pREP1-CYP4Z1
h- ura4.dl18::pCAD1-
CPR/pREP1-CYP2D6
h- ura4.dl18::pCAD1-
CPR/pREP1-CYP2B6
h- ura4.dl18::pCAD1-
CPR/pREP1-CYP2C19
h- ura4.dl18::pCAD1-
CPR/pREP1-CYP3A4
h- ura4.dl18::pCAD1-
CPR/pREP1-CYP2C9
h- ura4.dl18::pCAD1-
CPR/pNMT1-CYP17
h- ura4.dl18::pCAD1-
CPR/pNMT1-CYP21
h- ura4.dl18::pCAD1-
CPR/pREP1-hCYP3A7*2
h- ura4.dl18::pINT5-
CYP11B2
Myristic acid,
lauric acid
Dextromethorphan
CAD64
CAD65
CAD66
CAD67
CAD68
CAD71
CAD75
INA1
Peters et al., 2009a
Bupropion
Peters et al., 2009b
Mephenytoin
Dragan et al., submitted
Dragan et al., submitted
Dragan et al., submitted
Dragan et al., manuscript in preparation
Dragan et al., manuscript in preparation
Neunzig et al., submitted
Bureik et al., 2002
Midazolam,
testosterone
Tolbutamide
Progesterone
Progesterone, 17a-
OH-progesterone
DHEA, testosterone
MB164
SZ1
Deoxycorticosterone
Deoxycortisol
h- ura4.dl18::pCAD1-
CYP11B1
CYP11B1
Dragan et al., 2005
EMM culture grown for 3 days in the absence of thiamine to sta-
tionary phase. After a biomass growth phase of 40 h, 150 mg of
substrate dissolved in 3.0 ml ethanol was added. Another 150 mg
of substrate was added after 65 h to yield an end concentration of
Cytochrome P450 screening biotransformation
assays
Biotransformation assays with 17,17-dimethyl-18-norandrosta-
1,4,13-trien-3-one using the recombinant fission yeast strains shown
in Table 2 were started with a cell density of approximately 2=108
cells/ml and incubation was carried out in 96-well microtiter plates
at a shaking velocity of 150 rpm for 24 h at 308C. The substrate
approximately 200 mM. The total fermentation runtime was 132 h.
During the first 40 h (biomass growth phase) feeding was carried
out according to Jansen with minor modifications (Jansen et al.,
2006). One liter of the used feed medium contained 1 g Na2HPO4,
20 g (NH4)2SO4, 5 g KH2PO4, 4 g MgSO4, 150 g glucose, 20 ml
salt stock solution (Moreno et al., 1991), 1 ml vitamin stock solution
(Moreno et al., 1991) and 100 ml mineral stock solution (Moreno
et al., 1991). The glucose concentration was maintained at a value
between 0.1 and 1 g/l. After 40 h, the glucose concentration was
increased to a range between 2 and 8 g/l (biotransformation phase)
by manually adding an adequate volume of an aqueous 60% glucose
containing solution (w/v). Samples were frequently taken and ana-
lyzed for the determination of glucose concentration, biomass dry
weight as well as product formation. Steroids were extracted from
the fermentation broth with equal volumes of ethyl acetate and
evaporated using a rotary evaporator (Bu¨chi, Flawil, Switzerland).
concentration was 250 mM. All steroids were extracted from the cell
suspension twice with equal volumes of ethyl acetate and evapo-
rated. Steroids were then derivatized to per-TMS products with an
incubation at 608C for at least 15 min in a glass test tube in 100 ml
of a mixture of MSTFA, ammonium iodide and ethanethiol
(100:0.2:0.3, v/w/v). GC-MS analyses were performed on an Agi-
lent 6890/ 5973 instrument (Waldbronn, Germany) equipped with
an HP Ultra1 GC column (length 17 m, i.d. 0.2 mm, film thickness
0.11 mm) as previously described (Scha¨nzer et al., 2006).
The functionality of all P450 expressing fission yeast strains was
confirmed using the known standard substrates (Table 1) at a con-
centration of 1 mM.
Fermentation conditions
Product purification
Fed-batch cultivation was performed at 308C in a 5 l stirred-tank
fermenter (Biostat, Braun, Melsungen, Germany) at a stirrer speed
of 400 rpm. The start volume of the culture was 2.6 l and the
medium used for fermentation was DM1. The pH was continuously
measured by a pH electrode (Easyferm Plus K8, Hamilton, NV,
USA) and automatically kept constant at pH 5.5 by addition of
5 N NaOH. The fermenter was flushed with air at a flow rate of 3
l/min. The dissolved oxygen concentration was monitored continu-
ously with a pO2 measuring system (Oxyferm, Hamilton, NV, USA).
The fermentation medium was inoculated with biomass from a 2 l
Purification of the product was accomplished in three steps. First,
from the extraction residue the remaining substrate was removed
with the n-pentane layer via liquid-liquid extraction from a solution
in methanol:H2O (4:1; v/v). Subsequently, the hydroxylated prod-
ucts were extracted with n-pentane from a mixture of metha-
nol:aqueous KOH (1:9; v/v). Final purification was performed by
HPLC using a semi-preparative column from Macherey-Nagel
(Du¨ren, Germany) wNucleosil N(CH3)2, 10=250 mmx.
The preparative HPLC system consisted of a Hewlett Packard
(HP) 1050 series injector (Agilent Technologies, AT, Waldbronn,
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126 A. Zo¨ llner et al.
Germany) equipped with a 1.0 ml loop, an AT 1200 series G1361A
preparative pump, and an AT 1100 series diode array detector. The
product was isocratically eluted from the column with a flow of
5 ml/min using n-hexane:2-propanol (95:5) as solvent. Fractions
containing the desired metabolite were collected manually. The puri-
ty of the eluted products was analyzed via GC-MS as described
previously.
Guengerich, F.P. (2005). Human cytochrome P450 enzymes. In:
Cytochrome P450 – Structure, Mechanism, and Biochemistry,
P.R. Ortiz de Montellano, ed. (New York, USA: Kluwer Aca-
demic/Plenum Publishers), pp. 377–530.
Hannemann, F., Bichet, A., Ewen, K.M., and Bernhardt, R. (2007).
Cytochrome P450 systems – biological variations of electron
transport chains. Biochim. Biophys. Acta 1770, 330–334.
Ingelman-Sundberg, M., Sim, S.C., Gomez, A., and Rodriguez-
Antona, C. (2007). Influence of cytochrome P450 polymor-
phisms on drug therapies: pharmacogenetic, pharmacoepigenetic
and clinical aspects. Pharmacol. Ther. 116, 496–526.
Isin, E.M. and Guengerich, F.P. (2007). Complex reactions catalyzed
by cytochrome P450 enzymes. Biochim. Biophys. Acta 1770,
314–329.
NMR analysis
The nuclear magnetic resonance (NMR) analyses were performed
in CDCl3 (;4 m
M
solute) at 600 MHz (1H NMR) and 150 MHz
(13C NMR) on a Bruker Avance II 600 instrument (Bruker, Rhein-
1
stetten, Germany) at 298 K. The spectra were recorded as H; H,H
COSY; APT; H,C HMQC; H,C HMBC and NOESY using tetra-
methylsilane as internal standard.
Jansen, M.L.A., Krook, D.J.J., De Graaf, K., van Dijken, J.P., Pronk,
J.T., and de Winde, J.H. (2006). Physiological characterization
and fed-batch production of an extracellular maltase of Schizo-
saccharomyces pombe CBS 356. FEMS Yeast Res. 6, 888–901.
Kayes-Wandover, K.M. and White, P.C. (2000). Steroidogenic
enzyme gene expression in the human heart. J. Clin. Endocrinol.
Metab. 85, 2519–2525.
Acknowledgments
The authors would like to thank Dr. Susanne Zo¨llner for helpful
discussions and proof reading of the manuscript as well as Andrea
E. Schwaninger for assistance with the prep-HPLC. The Deutsche
Bundesstiftung Umwelt (DBU), the German Federal Ministry of the
Interior and the Manfred Donike Institute for Doping Analyses, e.V.,
Cologne, are acknowledged for financial support.
Korzekwa, K.R., Krishnamachary, N., Shou, M., Ogai, A., Parise,
R.A., Rettie, A.E., Gonzalez, F.J., and Tracy, T.S. (1998). Eval-
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models: evidence that multiple substrates can simultaneously
bind to cytochrome P450 active sites. Biochemistry 37,
4137–4147.
Lin, Y., Lu, P., Tang, C., Mei, Q., Sandig, G., Rodrigues, A.D.,
Rushmore, T.H., and Shou, M. (2001). Substrate inhibition kinet-
ics for cytochrome P450-catalyzed reactions. Drug Metab. Dis-
pos. 29, 368–374.
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Received August 5, 2009; accepted September 16, 2009
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