Doping test results dependent on genotype of uridine diphospho-glucuronosyl transferase 2B17, the major enzyme for testosterone glucuronidation.
ABSTRACT Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. The large variation in testosterone glucuronide (TG) excretion and its strong association with a deletion polymorphism in the uridine diphospho-glucuronosyl transferase (UGT) 2B17 gene challenge the accuracy of the T/E ratio test.
Our objective was to investigate whether genotype-based cutoff values will improve the sensitivity and specificity of the test.
This was an open three-armed comparative study.
A total of 55 healthy male volunteers with either two, one, or no allele [insertion/insertion, insertion/deletion, or deletion/deletion (del/del)] of the UGT2B17 gene was included in the study. Intervention: A single im dose of 500 mg testosterone enanthate was administered.
Urinary excretion of TG after dose and the T/E ratio during 15 d were calculated.
The degree and rate of increase in the TG excretion rate were highly dependent on the UGT2B17 genotype with a 20-fold higher average maximum increase in the insertion/insertion group compared with the del/del group. Of the del/del subjects, 40% never reached the T/E ratio of 4.0 on any of the 15 d after the dose. When differentiated cutoff levels for the del/del (1.0) and the other genotypes (6.0) were applied, the sensitivity increased substantially for the del/del group, and false positives in the other genotypes were eliminated.
Consideration of the genetic variation in disposition of androgens will improve the sensitivity and specificity of the testosterone doping test. This is of interest not only for combating androgen doping in sports, but also for detecting and preventing androgen abuse in society.
- SourceAvailable from: Jenny J Schulze[Show abstract] [Hide abstract]
ABSTRACT: The steroid module of the Athlete Biological Passport, the newest innovation in doping testing, is currently being finalized for implementation. Several factors, other than doping, can affect the longitudinal steroid profile. In this study, we investigated the effect of hormonal contraceptives (HC) as well as the effect of three polymorphisms on female steroid profiles in relation to doping controls. The study population consisted of 79 female elite athletes between the ages of 18 and 45. HC were used by 32% of the subjects. A full urinary steroid profile was obtained using World Anti-Doping Agency accredited methods. In addition all subjects were genotyped for copy number variation of UGT2B17 and SNPs in UGT2B7 and CYP17. Subjects using HC excreted 40% less epitestosterone as compared to non-users (p = 0.005) but showed no difference in testosterone excretion. When removing individuals homozygous for the deletion in UGT2B17, the testosterone to epitestosterone (T/E) ratio was 29% higher in the HC group (p = 0.016). In agreement with previous findings in men, copy number variation of UGT2B17 had significant effect on female urinary testosterone excretion and therefore also the T/E ratio. Subjects homozygous for the T allele of CYP17 showed a lower urinary epitestosterone concentration than the other CYP17 genotypes. It is of great importance that the athlete's steroidal passport can compensate for all possible normal variability in steroid profiles from women. Therefore, considering the large impact of HC on female steroid profiles, we suggest that the use of HC should be a mandatory question on the doping control form.Frontiers in Endocrinology 01/2014; 5:50.
- [Show abstract] [Hide abstract]
ABSTRACT: Concern for the health of athletes and integrity of sport resulted in the banning of specific substances although many years passed before analytical testing took place. Soon doping control programmes became synonymous with urine tests and adverse analytical findings. This system has its limits due to the detection window of prohibited substances, the timing of sample collections and the sophistication of some doping regimens. There have been a number of situations where these limits were demonstrated by athletes who proclaimed innocence based on passing their analytical tests only to later confess to doping. New strategies were called for to protect clean athletes. In the current World Anti-Doping Code, there are eight means to an Anti-Doping Rule Violation (ADRV). Article 2.2 states that the use of a prohibited substance may be established by any reliable means including witness statements, documentary evidence or evaluations of longitudinal profiling. In 2006, the World Anti-Doping Agency (WADA) with the support of some International Federations (IFs) gathered a group of experts to develop a harmonised programme on longitudinal profiling, or serial analysis of indirect biomarkers of doping, that was both scientifically and legally robust. This culminated in the WADA Athlete Biological Passport (ABP) Operating Guidelines and Technical Documents, published in 2009. The ABP is a paradigm that infers the use of prohibited substance (or method) by the monitoring of discriminant biomarkers over time. The haematological module detects blood manipulation by the use of erythropoietic stimulating agents or via blood transfusions. The steroidal module aims to identify endogenous anabolic androgenic steroids when administered exogenously and other indirect steroid doping substances or methods. Other ABP modules (endocrine, 'omics') are being developed. The term passport, first coined in 2000, is now defined in the ABP Guidelines as the longitudinal profile and all other relevant information including training, competitions and information derived from investigations. In the 2015 World Anti-Doping Code, investigations or enquiries gathered from other sources will play an even more prominent role.British journal of sports medicine 03/2014; · 3.67 Impact Factor
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ABSTRACT: In the fight against doping, steroid profiling is a powerful tool to detect drug misuse with endogenous anabolic androgenic steroids. To establish sensitive and reliable models, the factors influencing profiling should be recognised. We performed an extensive literature review of the multiple factors that could influence the quantitative levels and ratios of endogenous steroids in urine matrix. For a comprehensive and scientific evaluation of the urinary steroid profile, it is necessary to define the target analytes as well as testosterone metabolism. The two main confounding factors, that is, endogenous and exogenous factors, are detailed to show the complex process of quantifying the steroid profile within WADA-accredited laboratories. Technical aspects are also discussed as they could have a significant impact on the steroid profile, and thus the steroid module of the athlete biological passport (ABP). The different factors impacting the major components of the steroid profile must be understood to ensure scientifically sound interpretation through the Bayesian model of the ABP. Not only should the statistical data be considered but also the experts in the field must be consulted for successful implementation of the steroidal module.British journal of sports medicine 05/2014; 48(10):848-55. · 3.67 Impact Factor
Doping Test Results Dependent on Genotype of
Uridine Diphospho-Glucuronosyl Transferase 2B17,
the Major Enzyme for Testosterone Glucuronidation
Jenny Jakobsson Schulze, Jonas Lundmark, Mats Garle, Ilona Skilving, Lena Ekstro ¨m, and
Department of Laboratory Medicine, Karolinska Institutet at Division of Clinical Pharmacology, Karolinska University Hospital, SE-141 86
Context: Testosterone abuse is conventionally assessed by the urinary testosterone/epitestoster-
one (T/E) ratio, levels above 4.0 being considered suspicious. The large variation in testosterone
glucuronide (TG) excretion and its strong association with a deletion polymorphism in the uridine
diphospho-glucuronosyl transferase (UGT) 2B17 gene challenge the accuracy of the T/E ratio test.
sensitivity and specificity of the test.
Design: This was an open three-armed comparative study.
Participants: A total of 55 healthy male volunteers with either two, one, or no allele [insertion/
insertion, insertion/deletion, or deletion/deletion (del/del)] of the UGT2B17 gene was included in
Intervention: A single im dose of 500 mg testosterone enanthate was administered.
Main Outcome Measures: Urinary excretion of TG after dose and the T/E ratio during 15 d were
Results: The degree and rate of increase in the TG excretion rate were highly dependent on the
UGT2B17 genotype with a 20-fold higher average maximum increase in the insertion/insertion
group compared with the del/del group. Of the del/del subjects, 40% never reached the T/E ratio
of 4.0 on any of the 15 d after the dose. When differentiated cutoff levels for the del/del (1.0) and
and false positives in the other genotypes were eliminated.
Conclusions: Consideration of the genetic variation in disposition of androgens will improve the
Endocrinol Metab 93: 2500–2506, 2008)
cades (1), primarily for androgen replacement therapy in men
with androgen deficiency. Over the recent decades, testosterone
mid-1930s. It has been clinically used for nearly seven de-
and other androgens have been increasingly abused for muscle
study showed that power lifters with current or previous abuse
Printed in U.S.A.
Copyright © 2008 by The Endocrine Society
doi: 10.1210/jc.2008-0218 Received January 28, 2008. Accepted March 5, 2008.
First Published Online March 11, 2008
For editorial see page 2469
Abbreviations: AUC, Area under the curve; CI, confidence interval; cr, creatinine; Ct, cycle
threshold; del/del, deletion/deletion; DHT, dihydrotestosterone; EG, epitestosterone gluc-
uronide; ins/ins, insertion/insertion; ins/del, insertion/deletion; RQ, relative quantification;
T/E, testosterone/epitestosterone; TG, testosterone glucuronide; UGT, uridine diphospho-
O R I G I N A LA R T I C L E
E n d o c r i n e C a r e
jcem.endojournals.orgJ Clin Endocrinol Metab. July 2008, 93(7):2500–2506
without any exposure to anabolic steroid (3). It is possible that
the previous use of anabolic steroids may improve physical per-
formance for many years after withdrawal.
The World Anti-Doping Agency standardizes the rules and
regulations governing antidoping in elite sports internationally.
Anabolic compounds are the most frequently detected agents,
accounting for about 43% of positive results in 2005. Among
these testosterone, nandrolone and stanozolol were predomi-
Testosterone is excreted mainly as glucuronide conjugates
after metabolism by uridine diphospho-glucuronosyl trans-
ferases (UGTs). It is well established that UGT2B7, UGT2B15,
and UGT2B17 are the principal catalysts of the glucuronidation
of androgens and their metabolites in the human (4). Testoster-
UGT2B15 (5). The main androgen substrate of UGT2B15 is
UGT2B15 (6), but its substrate specificity is broader (5).
UGT2B7 has been shown to have the capacity to conjugate ep-
ples. Measuring only urinary testosterone glucuronide (TG) to de-
tect testosterone abuse is not adequate because of large interindi-
concentration. The nearly constant ratio of urinary TG to epites-
Medical Commission of the International Olympic Committee in-
troduced this value as a criterion for testosterone abuse. Ratios
above six should be considered suspicious, and the person con-
therefore, have lower T/E ratios, thus increasing the risk of false-
negative doping test results (12, 13). As a corollary the cutoff limit
was lowered to four in 2004.
We demonstrated that a deletion polymorphism in the gene
coding for UGT2B17 (14) is strongly associated with TG levels
in urine (15). All subjects devoid of the gene had a T/E ratio less
than 0.4 (15, 16). This polymorphism was considerably more
common in a Korean Asian than in a Swedish Caucasian pop-
Given this background we decided to monitor the testoster-
genotypes after testosterone administration. The aim was to in-
vestigate whether it is possible to increase the sensitivity and
For this purpose 14–24 healthy volunteers each with two inser-
tion/insertion (ins/ins), one insertion/deletion (ins/del), or zero
testosterone, and the urinary excretion of testosterone and EGs
suggest that urine analyses with combined genetic tests of the
UGT2B17 gene considerably improve the sensitivity and spec-
ificity of the T/E test.
Subjects and Methods
Subjects and design
fill the predetermined number of approximately 20 subjects in each of the
three different genotype panels. Among the 145 genotyped subjects, 15%
were homozygous for the gene deletion (del/del), 52% were heterozygous
originated from different ethnicities, the genotype frequencies are not rep-
14 ins/ins participants completed the study.
Study population characteristics are presented in Table 1. All par-
ticipants underwent a medical examination, including laboratory tests,
not interfere with the synthesis, metabolism, and excretion of steroids
illegal drugs, anabolic androgenic steroids, HIV, and hepatitis B or C
of any organization belonging to the Swedish Sports Confederation, or
had had a malignancy within the past 5 yr or an allergy to the study
substance. All participants gave informed consent consistent with the
approval of the Ethics Review Board.
Two individuals were excluded before the start of the study due to
pathological laboratory tests, two due to positive virological tests, and
one due to positive screening for illegal drugs. One subject was enrolled
and given testosterone but was later excluded due to treatment with a
substance that could interfere with analysis. The rest of the genotyped
subjects that did not participate were either dropouts (n ? 4) or did not
match the right genotype panel. The participants were given 500 mg
testosterone enanthate in castor oil as a single im dose of Testoviron
alent to 360 mg testosterone. Before administration (d 0), urine samples
and 15. All samples were collected between 0700 and 1100 h. Adverse
administration of testosterone. No major adverse drug reactions were
registered. No follow-up was needed. The study was conducted accord-
Study population characteristics at screening
del/del (n ? 17)
ins/del (n ? 24)
ins/ins (n ? 14)
27.2 ? 4.0
32.0 ? 7.7
28.7 ? 7.0
179 ? 7.5
180 ? 6.6
181 ? 5.4
75.9 ? 8.4
79.6 ? 9.1
78.6 ? 6.0
23.8 ? 2.6
24.6 ? 2.9
24.0 ? 2.1
Values are mean ? SD. BMI, Body mass index.
J Clin Endocrinol Metab, July 2008, 93(7):2500–2506jcem.endojournals.org
Blood and urine samples
Venous blood was obtained from the cubital vein and collected in
EDTA tubes for DNA extraction. The urine samples were collected and
kept refrigerated for a maximum 48 h and then frozen at ?20C.
Copy number analysis of UGT2B17
The copy number of the UGT2B17 gene was assessed by real-time
PCR analysis. Ten nanograms of genomic DNA were used in each re-
action together with 2? TaqMan Universal Master Mix (Applied Bio-
an exon 6 specific probe (VIC-CAGTCTTCTGGATTGAGTTT-MGB).
Expression of albumin was quantified as an endogenous control as de-
on the same plate. The probe concentrations were 100 nM in each assay,
and the primer concentrations were 900 and 600 nM for the UGT2B17
and albumin-specific reactions, respectively. The PCR profile consisted
of an initial denaturation step at 95 C for 10 min, followed by 40 cycles
of 92 C for 15 sec and 60 C for 1 min. The effect of DNA concentration
on PCR efficiency was determined using a control DNA in a dilution
series of 20, 15, 10, 7.5, 5, and 2.5 ng/reaction. A known ins/del sample
was chosen as a calibrator. It was set to one, and the relative quantifi-
cation (RQ) was calculated using the ??CT method (18).
Urinary unconjugated steroids and steroid glucuronides were ana-
lyzed at the Doping Laboratory of the department. Aliquots of 2–8 ml
(depending upon the specific gravity of the urine sample) were comple-
mented with 1 ?g methyltestosterone as an internal standard. The un-
conjugated steroids were extracted directly with 5 ml tert-butyl methyl
ether. The glucuronidated steroids were hydrolyzed with ?-glucuroni-
dase from Escherichia coli (Roche Diagnostics, Mannheim, Germany)
[(pH 7.0) 50 C for 1 h] and extracted in 5 ml n-pentane [(pH 8.5) room
temperature for 10 min]. The organic phase was evaporated to dryness
under a stream of nitrogen. Samples were converted into enol-trimeth-
ylsilyl ether derivatives with N-methyl-N-trimethylsilyltrifluoroacet-
amide (Macherey-Nagel, Du ¨ren, Germany) and ammonium iodide as
described previously (19).
Urinary steroids were determined using combined gas chromatogra-
ogies, Inc., Palo Alto, CA) with the Single Ion Monitoring mode (19).
one-point calibration with a mixture of authentic standard materials
analyzed with every batch of samples. In addition to testosterone and
epitestosterone, the androgen metabolites 5?-androstane-3?,17?-diol,
androsterone, and etiocholanolone were measured. Interference with
testosterone in the assay from, e.g. certain drugs, was not found in any
of the samples. The day-to-day variation of the instrument was mini-
mized using the mixture of authentic standards analyzed with every
for all steroids analyzed were less than 7 and 8%, respectively.
dividing the concentration values by the urinary creatinine (cr) concen-
tration. All urinary values are expressed as the unconjugated (typically
less than 3% of the glucuronide fraction) plus the glucuronide conju-
gated fraction after correction for cr, if not specified otherwise.
The areas under the curves (AUCs) of the different urinary steroids
were calculated using the trapezoidal rule.
Statistical analyses were performed by Kruskal-Wallis analysis, fol-
lowed by Dunn’s multiple comparison post hoc test with P ? 0.05 re-
garded as significant.
UGT2B17 copy number assay
When the cycle threshold (Ct) values of UGT2B17 and al-
bumin were plotted vs. log DNA concentration, the PCR effi-
ciency for the UGT2B17 and albumin reaction was similar, 97
and 95%, respectively, and the difference between the slopes
(Cttarget? Ctreference) was less than 0.1, showing that the ?Ct
signal was observed were considered as homozygous for the de-
letion allele (del/del). Individuals with one allele (ins/del) had a
mean RQ value of 1.04 (range 0.89–1.28), and individuals with
2.62). There was no overlap between the groups demonstrating
an unequivocal interpretation of genotyping results.
jects (two ins/ins, one ins/del, and four del/del) before and after
of the glucuronidated fraction without differences between the
genotypes. It was concluded that excretion of unconjugated tes-
tosterone after testosterone administration is only a minor elim-
ination pathway even for the del/del subjects. Therefore, the un-
conjugated fraction was not isolated and analyzed separately in
the remaining subjects.
Baseline urinary steroids
The average baseline urinary unconjugated and glucu-
ronidated testosterone (TG) and epitestosterone (EG) concen-
trations and T/E ratios are presented in Table 2. The TG levels
groups (P ? 0.001). There was no statistically significant differ-
ence in the EG levels.
The T/E ratio in the del/del group also differed significantly
(P ? 0.001). The ratios in the ins/ins and ins/del groups did not
differ significantly from each other.
Baseline urinary androgen glucuronide levels
del/del (n ? 17)
ins/del (n ? 24)
ins/ins (n ? 14)
2.8 (1.2- 4.5)
The values are given as the mean with the 95% CI within parentheses, or as the ratio between TG and EG levels.
aStatistical significance between the genotypes (P ? 0.001).
Schulze et al. UGT2B17 and Testosterone DopingJ Clin Endocrinol Metab, July 2008, 93(7):2500–2506
There were no significant differences between the geno-
types in the urinary concentrations of glucuronides of andro-
sterone, etiocholanolone, or 5?-androstane-3?,17?-diol,
which are the major final metabolites of testosterone and di-
hydrotestosterone (DHT) (data not shown). These three me-
tabolites are, in addition to UGT2B17, also conjugated by
UGT2B7 and UGT2B15.
Urinary steroid profile after testosterone administration
testosterone dose is shown in Fig. 1A. The maximum average
increase in TG excretion after dose was 2.0 ng/?mol cr [95%
confidence interval (CI) 1.4–2.6] in the del/del group, 18.6 ng/
?mol cr (95% CI 12.9–24.4) in the ins/del group, and 41.8
ng/?mol cr (95% CI 27.9–55.6) in the ins/ins group (Fig. 1B).
FIG. 1. A, Urinary TG excretion (ng/?mol cr) for 15 d in all subjects of the UGT2B17 ins/ins, ins/del, and del/del groups after an im injection of 500 mg
testosterone enanthate, equivalent to 360 mg testosterone, on d 0. Note that the y-axes have different scales. B, Average urinary TG excretion (ng/?mol
cr) for 15 d in the different genotype groups after an im injection of 500 mg testosterone enanthate, equivalent to 360 mg testosterone, on d 0. Vertical
bars denote 95% CIs. C, Average urinary EG excretion (ng/?mol cr) for 15 d in the different genotype groups after an im injection of 500 mg testosterone
enanthate, equivalent to 360 mg testosterone, on d 0. Vertical bars denote 95% CIs.
J Clin Endocrinol Metab, July 2008, 93(7):2500–2506jcem.endojournals.org
in the del/del group, 162 ng/?mol cr (95% CI 129–195) ? day
in the ins/del group, and 294 ng/?mol cr (95% CI 236–351) ?
the del/del group and the other two groups (P ? 0.001), and
between the ins/del and the ins/ins group (P ? 0.05).
The excretion of epitestosterone glucuronide decreased to
levels close to zero for all subjects without any statistically sig-
nificant differences between the genotypes (Fig. 1C).
The average urinary T/E ratio increased from 0.14 (95% CI
0.11–0.18) to 5.3 (95% CI 4.1–6.5) in the del/del group, from
1.4 (95% CI 1.1–1.6) to 50.4 (95% CI 39.1–61.6) in the ins/del
group, and from 2.3 (95% CI 1.7–2.9) to 100 (95% CI 70.8–
130) in the ins/ins group (Fig. 2).
There were no significant differences between the individual
genotypes in urinary excretion of the major testosterone and
DHT metabolites etiocholanolone-G and androsterone-G (data
not shown). The del/del group had a lower AUC of the major
DHT metabolite 5?-androstane-3?,17?-diol-G (172 ng/?mol
cr ? day; 95% CI, 204–293) (P ? 0.05).
Sensitivity of the test after a single testosterone dose
The sensitivity of the current testosterone doping T/E test is
shown in Fig. 3, left panel, and Table 3. When the ratio is set to
one for the del/del group and six for the ins/del and ins/ins
groups, the sensitivity increased substantially for the del/del
group, and the number of false-positive doping tests was elimi-
nated for the ins/ins group (Fig. 3, right panel, and Table 3).
in TG excretion after a single im testosterone dose are highly
dependent on the genotype of the major testosterone glucu-
ronidating UGT2B17 enzyme gene. Our findings have implica-
analysis in doping tests. Genotyping as a complement to the
conventional urine analysis would improve the sensitivity of the
test by introduction of genotype-based differentiated cutoff
There were large differences in TG excretion after testoster-
we show that 40% of the subjects without the UGT2B17 gene
never reached the T/E cutoff ratio of 4.0 on any of the 15 d after
for the del/del group (5.3) was reached on d 9. The other two
groups reached their highest average ratio earlier, on d 7 (50.4)
and d 6 (100) for the ins/del and ins/ins group, respectively
Previous observations have shown that individuals of Asian
origin excrete lower amounts of TG than other populations (13,
20). Recently, we showed that a large part of the differences in
TG excretion could be explained by genetic variation of the
on genotypes, not ethnicity. The del/del genotype is much more
common in Asian populations (66.7%) than in Caucasians
of the urinary TG levels between the del/del group and the other
two genotypes. In total we have now analyzed urine samples
from 100 del/del individuals in this and two other studies (15,
16). All of them had TG levels less than 1 ng/?mol cr. The low
basal TG levels indicate that there are other UGT enzymes that
catalyze the glucuronidation, most likely
UGT2B15 (5). The TG levels did increase
after injection of testosterone enanthate,
ins/ins group. This clearly shows the inad-
equacy of using the same cutoff level for all
Because UGT2B15 may contribute to
the low levels of urinary TGs in UGT2B17
del/del subjects, one could speculate that
polymorphisms in this gene may also affect
the T/E ratio. There is a G to T polymor-
FIG. 2. Average urinary T/E ratios for 15 d in the different genotype
groups after an im injection of 500 mg testosterone enanthate,
equivalent to 360 mg testosterone, on d 0. Vertical bars denote 95% CIs.
FIG. 3. Sensitivity of the testosterone doping test using a cutoff ratio of four (left panel) or cutoff
ratios of six for the ins/ins and the ins/del groups and one for the del/del group (right panel). A
single im dose of 500 mg testosterone enanthate was administered in 14 ins/ins, 24 ins/del, and 17
del/del subjects on d 0, and the urinary T/E ratios were measured for 15 d.
Schulze et al. UGT2B17 and Testosterone Doping J Clin Endocrinol Metab, July 2008, 93(7):2500–2506
amino acid change at position 85 (21). However, this single nu-
cleotide polymorphism does not seem to change the basal T/E
ratios (16) or affect the T/E ratio in individuals devoid of the
lished results). Another polymorphism that may affect the T/E
ratio is the H268Y polymorphism (22) in the epitestosterone-
conjugating UGT2B7 enzyme (7). However, we have recently
in addition to the UGT2B17 deletion remains to be studied.
Exogenous testosterone is known to decrease the urinary ex-
cretion rate of EG due to suppression of the secretion of LH
groups after the testosterone injection. There were large inter-
individual differences, but 6 d after the testosterone administra-
leading to even higher increases of T/E ratios.
Today, a T/E ratio cutoff limit of four gives cause for suspi-
cion of testosterone doping. This test would misjudge over 40%
the highest after a single dose of testosterone (Fig. 3, left panel).
The genetic variability within and between ethnic groups is a
On the contrary, in the ins/ins group, 14% had baseline T/E
ratios above four. In our previous study (15) of a population
sample of 122 young men, this limit would give a false-positive
rate of 9%. False-positive results are not only of concern for the
legal rights of the sportsman; they also yield an extra workload
for the doping laboratories.
Baseline values of T/E ratios in the del/del subjects never ex-
ceeded 0.4. We simulated a differentiated cutoff level for the
del/del (1.0) and the other genotypes (6.0), and found that at
(Fig. 3, right panel) in our experimental setting.
Testosterone can also undergo conjugation with sulfate be-
fore elimination. The urinary fraction of sulfate-conjugated tes-
in a reference population of 45 males aged 17–50 yr (27). Our
study was not designed to investigate other excretion pathways,
but it cannot be excluded that del/del subjects eliminate a larger
fraction of sulfate-conjugated testosterone to compensate for
their compromised capacity to glucuronidate testosterone.
The determination of the13C/12C ratio of selected steroids
(isotope ratio mass spectrometry analysis) provides the possibil-
ity to distinguish between pharmaceutical and natural testoster-
one because exogenous compounds contain less13C than their
endogenous homologs (28). However, the analytical facilities
and costs required preclude any routine use of this methodology
for screening in the antidoping testing. Therefore, its major use
is to confirm suspected doping in samples with T/E ratios equal
or greater than 4.0.
From Fig. 1A it seems that the TG excretion rate could be
divided into at least two groups. In one group the subjects
reached the peak urinary TG levels within 24 h, compared with
in the “slow rise” group compared with the “fast rise” group.
The reason for the distinct division into two parts in the testos-
didate genes include esterases that hydrolyze the ester in the
testosterone enanthate, but the particular enzyme involved in
this cleavage has not been identified. The reason for this TG
excretion pattern is of interest to study further because it may
influence both the biological effect of testosterone treatment as
well as the outcome of the doping test.
A logical follow-up of our test program is to investigate
whether the effects of testosterone are different in individuals
with different genotypes. We have recently shown that the base-
line serum testosterone levels are not associated with the
UGT2B17 polymorphism (16). However, the serum levels of
testosterone in the different UGT2B17 genotypes after exoge-
nous testosterone administration remain to be studied.
In summary, consideration of the genetic variation in andro-
gen disposition is important in featuring the androgen urinary
excretion profile, whether this is made for research purposes or
We thank Birgitta Ask for technical assistance.
Address all correspondence and requests for reprints to: Jenny J.
Schulze, Ph.D., Clinical Pharmacology C1:68, Karolinska University
Hospital, Huddinge, 141 86 Stockholm, Sweden. E-mail: jenny.
This study was supported by the World Anti-Doping Agency, the
Swedish Cancer Society, and the Cancer Society in Stockholm.
Disclosure Statement: The authors have nothing to disclose.
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Sensitivity of the test with a cutoff T/E ratio of 4.0 for all subjects, or a cutoff T/E ratio of 1.0 for del/del and 6.0 for
del/del subjects (%) ins/del subjects (%) ins/ins subjects (%)
Cutoff T/E ratio
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