Clinical Chemistry 46: 515-522, 2000;
(Clinical Chemistry. 2000;46:515-522.)
© 2000 American Association for Clinical Chemistry, Inc.
Oral Testosterone Administration Detected by Testosterone Glucuronidation Measured in Blood Spots Dried on Filter Paper
Shi-Hua Peng1,
Jordi Segura1,2,a,
Magí Farré1,3 and
Xavier de la Torre1,3
1
Institut Municipal d’Investigació Mèdica, E-08003 Barcelona, Spain.
2
Universitat Pompeu Fabra, Barcelona, Spain.
3
Universitat Autónoma de Barcelona, Barcelona,
Spain.
a Address correspondence to this author at: Drug Research Unit, Institut Municipal d’Investigació Mèdica (IMIM), Doctor Aiguader 80, E-08003 Barcelona, Spain. Fax 34-93-2213237; e-mail jsegura{at}imim.es
 |
Abstract
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Background: Blood sampling is not a common
practice for sports drug testing. Our aim was to investigate whether
dried blood spots on filter paper could be an alternative to plasma
samples for monitoring steroid profiles in dope testing.
Methods: We collected dried blood spots and plasma from six
healthy Caucasian subjects after an oral 120-mg dose of testosterone
undecanoate (TU). Nonconjugated testosterone, testosterone
glucuronide (TG), androsterone glucuronide (AG), and etiocholanolone
glucuronide (EtG) were measured by gas chromatography–mass
spectrometry in both matrices. 17
-Hydroxyprogesterone (17OHP) and
luteinizing hormone (LH) also were measured in the plasma samples. For
comparison, similar measurements were done on samples obtained from the
same subjects given 25 mg of testosterone propionate (TP) plus 110 mg
of testosterone enanthate (TE) intramuscularly after a wash-out
period.
Results: After oral TU intake, TG, AG, and EtG increased sharply,
whereas nonconjugated testosterone did not change significantly.
Results on dried blood spots correlated well with those on plasma. The
TG/testosterone ratio in blood or plasma was verified to be a
sensitive and specific marker (significantly increased for up to 8
h after intake; P <0.05) for oral TU intake but
not for intramuscular administration of TP plus TE. Little suppression
of plasma LH and 17OHP was observed after a single oral dose of TU.
One subject did not show a significant increase of blood TG after oral
TU intake.
Conclusions: The measurement of glucuronide conjugates in blood
and plasma samples is relevant for sports drug testing when analyzing
the steroid profile. Dried blood spots collected on filter paper are a
suitable alternative to plasma for detecting testosterone
abuse.
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Introduction
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In recent years, there has been an increase of doping with
"endogenous-like substances", such as testosterone and some of its
metabolic precursors. Discriminating the exogenous intake of these
substances from their endogenous origin constitutes a challenge in
today’s sports drug testing. Pharmaceutical testosterone preparations
are commercially available for oral, percutaneous, and intramuscular
administration. The International Olympic Committee criterion
for suspecting exogenous testosterone intake, a urinary concentration
ratio of testosterone glucuronide to epitestosterone glucuronide
(TG/EG)1
>6 (1), needs elaborate follow-up for definitive
decisions. There are a few subjects who have physiologically increased
urinary TG/EG ratios, whereas other individuals do not show a TG/EG
ratio >6 even after exogenous intake of testosterone
(2)(3). In addition, oral administration of
testosterone produces a high but brief increase in the urinary TG/EG
ratio (4), which probably would be difficult to detect (as
judged by the criterion TG/EG >6) as an indication of exogenous
testosterone intake when urine is collected after a sporting event
(which usually includes a 1-h delay plus an additional waiting period
for the final collection of urine). Apart from measurement of the
13C content of urinary testosterone metabolites
by isotope-ratio mass spectrometry (5), a theoretically
better approach might be to analyze one drop of blood collected
immediately after the sporting event. The sample may be obtained by
fingerprick or earprick, which currently are used for lactic acid and
other measurements. The relevant question is whether suitable
information from adequate markers can be obtained from such a small
amount of sample. In the present study, one 120-mg dose of testosterone
undecanoate (TU) was administrated orally to six healthy Caucasian
volunteers. Dried blood spots and plasma samples were collected and
studied for biological indicators of exogenous testosterone intake.
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Materials and Methods
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references, steroids, and chemicals
Testosterone, testosterone glucuronide (TG), androsterone,
androsterone glucuronide (AG), etiocholanolone, etiocholanolone
glucuronide (EtG), 17-hydroxyprogesterone (17OHP), and
Escherichia coli ß-glucuronidase were purchased from
Sigma.
Testosterone-[16,16,17]-d3
(T-D3) and
etiocholanolone-[2,2,4,4]-d4
(Et-D4) were kindly provided by the late Prof. M.
Donike (Deutsche Sporthochschule, Cologne, Germany).
N-Methyl-N-trimethylsilyl-trifluoroacetamide
was provided by Macherey-Nagel (Düren, Germany). Ammonium iodide
and 2-mercaptoethanol were obtained from Merck. All other reagents were
of analytical grade.
sample collection
Six healthy male Spanish volunteers (subjects 1–6; age, 27.2
± 2.1 years; weight, 73.4 ± 4.0 kg; height, 1.75 ±
0.03 m, mean ± SD) received a single oral 120-mg dose of TU
(Androxon, three 40-mg capsules; Organon). Venous blood (10 mL) was
collected from the antecubital vein before and after administration.
Sampling times were 0.5 h (-0.5 h) before administration, at
administration (0 h; 0900 in the morning), and 0.5, 1, 1.5, 2, 3, 4, 6,
8, 10, and 12 h after administration. Aliquots of 20 µL
of fresh blood were spotted onto filter paper (Whatman 41), and then
air dried, sealed in plastic bags, and stored at -20 °C until
analysis. The remaining heparinized whole blood was centrifuged for
preparing plasma samples.
To check the stability of steroids in blood spots dried on filter
paper, aliquots of blood spot samples (stored at -20 °C) from each
of the six volunteers obtained 1 h after the oral TU
administration were removed from the freezer and kept at room
temperature for 1 week. These aliquots were analyzed for steroid
profiles simultaneously with the corresponding aliquots stored at
-20 °C from the time of collection.
After a wash-out period of 
3 months, the same six subjects received
a single intramuscular dose of Testoviron Depot 100 [25 mg of
testosterone propionate (TP) plus 110 mg of testosterone enanthate
(TE); Schering AB]. Blood spots and plasma samples were collected and
stored as described above. The sampling times were on days -3 (0900),
-2 (0900), -1 (0900) before administration; at 0 h (0900), 6 h
(1500), and 12 h (2100) after administration (on day 0), on days 1 and
2 at 0 h (0900) and 8 h (1700) on days 3 and 4; and at 0 h (0900)
on days 5, 6, 7, 8, 9, 11, 13, 15, 17, and 19 after administration.
Approval for the above-described clinical trials was granted by the
local ethics committee (CEIC no. 94/467) and by the Spanish Health
Ministry (DGFPS no. 95/75).
sample preparation
Dried blood spots.
For the extraction of nonconjugated
steroids from blood spots on filter paper, two dried blood spots were
cut with scissors in small pieces into a tube, and 2.5 mL of sodium
phosphate buffer (0.2 mol/L, pH 7) was added.
T-D3 and Et-D4 were used as
internal standards at final concentrations of 0.9 and 5 µg/L,
respectively, in buffer. Immediately after the subsequent addition of
100 µL of 3 mol/L potassium hydroxide, the samples were
extracted twice with 5 mL of n-hexane:ethyl acetate (7:3, by volume),
using a rocking mixer for 20 min to extract nonconjugated steroids. It
has been verified that under these conditions there is no hydrolysis of
testosterone esters, which could increase the actual amount of
testosterone initially present in the sample. The organic phases were
pipetted out into a tube, mixed, and washed with 1 mL of 50
mL/L acetic acid and then with 1 mL of distilled water. After
the solvent was evaporated, the residue was kept in a desiccator
maintained at 60 °C and 60 kPa (phosphorous pentoxide was used as
desiccant, which was changed when formation of phosphoric acid was
quite notable) at least 30 min before derivatization (see below). For
the extraction of glucuronide-conjugated steroids, the extracted
potassium hydroxide-water phase indicated above was neutralized with 1
mol/L hydrochloric acid, incubated with 6 U of ß-glucuronidase at
37 °C overnight after a second addition of the same amounts of the
same (T-D3 and Et-D4)
internal standards, and then extracted with t-butyl methyl
ether after the aqueous solution was adjusted to approximately pH 10
with 50 g/L potassium carbonate. After the solvent was
evaporated, the residue was kept in a desiccator at least 30 min before
derivatization. The dried extracts were derivatized for gas
chromatography–mass spectrometry (GC-MS) analysis. Trimethylsilyl
(TMS) derivatives of steroids in both extracts were prepared by
dissolving the dried residues in 50 µL of a mixture containing 1 L of
N-methyl-N-trimethylsilyl-trifluoroacetamide, 2 g
of ammonium iodide, and 6 mL of 2-mercaptoethanol and heating at
60 °C for 30 min. Each solution (1–2 µL) was directly analyzed by
GC-MS. Endogenous steroids (testosterone, androsterone, and
etiocholanolone) were quantified using the responses of the target
compounds relative to the internal standards, calculated
from an extracted sample of distilled water containing 1.2 µg/L
testosterone, 20 µg/L androsterone, and 20 µg/L etiocholanolone
[extracted with hexane-ethyl acetate (7:3, by volume)].
Plasma.
A 1-mL plasma sample was diluted with 1.5 mL of sodium
phosphate buffer (0.2 mol/L, pH 7) and subjected to a procedure similar
to that for the analysis for urine in sports drug testing
(6), with modifications. Briefly, the diluted plasma sample
was loaded on a DetectabuseTM column pretreated
with methanol and water. The column was washed with water, and the
steroids were eluted with methanol. The solvent was evaporated, and the
residue was reconstituted in 1 mL of sodium phosphate buffer (0.2
mol/L, pH 7). Nonconjugated steroids (containing testosterone and
17OHP) were extracted with 5 mL of t-butyl methyl ether,
evaporated with nitrogen, and kept in a desiccator until
derivatization. The remaining aqueous phase (containing TG, AG, and
EtG) was then hydrolyzed with 6 U of ß-glucuronidase and extracted
using the same procedure that was used for the blood-spot samples.
Quantification of steroids (including 17OHP) in both fractions of
plasma was performed as for the blood spots samples, with modifications
that included a water calibration mixture containing 5 µg/L 17OHP,
12 µg/L testosterone, 200 µg/L androsterone and 200 µg/L
etiocholanolone, and monitoring the tris-TMS derivative of 17OHP in
the nonconjugated fraction extracted from plasma. Pooled plasma samples
and pooled plasma samples supplemented with testosterone, TG, AG, and
17OHP were run in each analytical batch as quality-control samples.
The intra- and interassay variabilities (CVs) were from 2.7% (for 12.7
nmol/L testosterone) to 15% (for 0.82 nmol/L TG). Accuracy
(relative error) was from -0.8% (for 29.6 nmol/L EtG) to -15.1%
(for 135 nmol/L AG).
Luteinizing hormone (LH) was analyzed by microparticle enzyme
immunoassay (Abbott) directly in plasma according to the
manufacturer’s instructions.
gc-ms analysis
A Hewlett-Packard 5890 II GC model fitted with a HP 7673A
autosampler was connected to a HP 5970 mass-selective detector. The
separation was carried out using a methyl silicone fused-silica
capillary column [HP Ultra-1; 17 m x 0.2 mm (i.d.); film
thickness, 0.11 µm] with the following oven temperature program:
initial temperature of 180 °C, ramped to 230 °C at a rate of
3.0 °C/min, then to 310 °C at a rate of 40 °C/min, and held
for 3 min. Helium was used as carrier gas with a flow rate of 0.8
mL/min (measured at 180 °C). The injector (operated in 10:1 split
mode) and the interface were maintained at 280 °C. The mass
spectrometer was operated in selected-ion monitoring
acquisition mode with one or more ions selected for each substance
(m/z 432, 434, and 434 for bis-TMS derivatives of
testosterone, androsterone and etiocholanolone, respectively;
m/z 435 and 438 for bis-TMS derivatives of
T-D3 and Et-D4,
respectively; m/z 546 for tris-TMS derivative of 17OHP).
statistical analysis
The concentrations in blood collected on filter paper and in
plasma, and their ratios (log-transformed) after administration of TU
were compared to their basal values by ANOVA for testing of significant
differences.
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Results
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blood steroid profile analyzed in dried blood spots
The nonconjugated testosterone measured in dried blood spots after
oral administration of TU did not change significantly. Instead TG,
which had an extremely low basal concentration in dried blood spots
(much lower than nonconjugated testosterone), increased substantially
after oral administration of TU in all of the subjects (Table 1
) except one (subject 3). Results for the abnormal subject are
discussed separately. Other main metabolites of testosterone, such as
AG and EtG, were also significantly increased in these five subjects.
Thus, hydrolysis of the dried blood sample plays an important role in
the interpretation of results. TG, AG, and EtG increased significantly
(compared with their basal concentrations) up to 8, 8, and 12 h,
respectively (Fig. 1
). Because of its extremely low basal concentration, TG showed
the highest relative increase. Accordingly, for this report, the
concept of steroid profile will refer specifically to those
concentrations of testosterone, TG, AG, and EtG.
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Table 1. Time course of blood steroid concentrations (mean ±
SE; n = 5) and TG/T ratio after a single oral 120-mg dose of
TU.
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Figure 1. Mean relative increases ([increase/basal concentration]
x100; n = 5) of TG, AG, and EtG obtained from blood spots dried
on filter paper after oral administration of 120 mg of TU.
y axis is in logarithmic scale.
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correlation with plasma steroid profile
The above-mentioned steroids and their glucuronides were analyzed
in plasma samples obtained at the same time. Similar patterns between
blood collected on filter paper and plasma were observed, with slightly
higher values obtained in blood (Fig. 2
).
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Figure 2. Mean concentrations (n = 5) of testosterone, TG, AG,
and EtG in blood spots and plasma after oral administration of 120 mg
of TU.
y axis is in logarithmic scale.  , testosterone in
dried blood spots;  , testosterone in plasma samples;  , TG in
dried blood spots;  , TG in plasma samples;  , AG in dried blood
spots;  , AG in plasma samples; •, EtG in dried blood spots;  ,
EtG in plasma samples. Conc., concentration.
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stability of steroid glucuronides in dried blood spots
It is known that androgenic anabolic steroids, such as
testosterone and 17OHP, are stable in dried blood spots
(7)(8). The stability of the glucuronides of
some steroids in dried blood spots stored for 1 week at room
temperature, compared with aliquots maintained in a freezer
(-20 °C), was verified (Fig. 3
). All four steroids measured (TG, testosterone, AG, and EtG)
were sufficiently stable during the study period. The mean
relative changes in TG, testosterone, AG, and EtG for the six subjects
after 1 week at room temperature were -5.9%, -2.8%, -6.9%,
and -4.8%, respectively.
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Figure 3. Stability of TG, nonconjugated testosterone, AG, and EtG
in blood spots dried on filter paper after oral administration of 120
mg of TU.
One aliquot was kept at -20 °C in freezer (); another aliquot
was kept at room temperature for 1 week (). T,
nonconjugated testosterone; sub, subject;
Conc., concentration.
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specificity of tg/testosterone as a marker of oral administration
To check whether the ratio of TG to testosterone (TG/T) in dried
blood spots could be a specific marker for oral vs intramuscular
administration of testosterone, dried blood-spot samples from the same
six subjects, who received intramuscular injections with a single
combined dose of 25 mg of TP plus 110 mg of TE were partially analyzed
for steroid profiles. In the first step, all of the dried blood spots
from one subject (subject 2) after intramuscular administration were
studied. Testosterone, AG, and EtG, but not TG, were clearly increased
after intramuscular administration. The highest increase of
nonconjugated testosterone was found in those blood spots on filter
paper collected between 1.25 and 2.25 days after intramuscular
administration of TP plus TE, which is in good agreement with the
pharmacokinetic profile known for the same type of preparation and
obtained by either RIA (9) or GC-MS (10). In the
next step, samples obtained 1 day before (-1 day), and 2.25 and 19
days after (+2.25 and +19 days) intramuscular administration from each
subject were selected for a more comprehensive analysis. For all the
six subjects, nonconjugated testosterone at +2.25 days was increased
(relative increases, 100–200%) compared with its basal concentration,
whereas no meaningful changes of TG were observed. However, clear
increases of AG and EtG were also observed in the blood collected 2.25
days after intramuscular dosing of testosterone esters. The results are
shown in Fig. 4
. The results obtained from corresponding plasma samples are
also presented. When comparing these results with the changes in
steroid profiles observed in blood after oral intake of testosterone,
testosterone glucuronidation in human blood appears to be a specific
marker for oral testosterone intake.
other markers (tg/lh, tg/17ohp, and tg/eg)
Recently, plasma T/LH and T/17OHP have been suggested as
sensitive markers of testosterone administration, based on the
observation that lower concentrations of plasma LH and 17OHP could
be induced by intramuscular administration of exogenous testosterone
(11)(12)(13). However, a single oral administration of TU seems
unlikely to cause significant suppression of LH secretion and
steroidogenesis. LH concentrations showed no meaningful changes during
024 h post administration, whereas 17OHP showed only a small
reduction. As shown in Table 2
, plasma TG/17OHP was affected, but mainly because of an
increase in TG rather than a decrease in 17OHP. In addition, plasma
TG/17OHP and TG/LH were significantly increased above their basal
concentrations until 10 and 6 h, respectively, post
administration. TG/EG in urine was >6 in all subjects but subject 3 in
the period 04 h, and in three subjects in the period 48 h post
administration (14).
outlier subject
One subject (subject 3; Table 3
) did not show significant changes of TG in plasma but did show
a transient (12 h) increase in nonconjugated testosterone after TU
administration. The AG and EtG concentrations in his dried blood spots
on filter paper did present remarkable increases. It has been verified
that his urinary TG/EG ratio remained <2 during the whole controlled
study (14).
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Discussion
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The clear increase of TG measured in dried blood spots of five of
six subjects after oral administration of TU appears to be an efficient
marker for exogenous intake of testosterone. Regarding the
extremely low basal concentration of TG and relative higher basal
concentration of testosterone, the TG/T ratio is a sensitive marker for
oral TU administration.
In the late 1970s, when oral TU preparations were studied as potential
new supplements for hypogonadal deficiency or fertility control,
clinical endocrinologists monitored extensively the effect of oral TU
on the plasma concentration of nonconjugated testosterone after oral TU
administration (15)(16)(17). It was reported that after
a single oral dose of TU, there were significant increases of
nonconjugated testosterone in both saliva and serum, as detected by RIA
(18)(19), which seems in apparent disagreement
with the absence of significant changes of nonconjugated testosterone
in plasma and dried blood spots observed in the present study by GC-MS.
It is feasible that some cross-reactivity (0.2%) (18) of
the RIA antiserum used in those previous studies for TU could produce
an apparent increase of nonconjugated testosterone, because the
unchanged TU is also extracted by the solvent extraction method used in
those studies. In fact, the existence of substantial amounts of TU in
plasma after oral TU administration is known (20)
and was confirmed in those subjects (14). The additional
thin-layer chromatographic clean-up of nonconjugated testosterone
preceding the RIA assay in some of these previous studies
(18), however, seems to preclude such an easy explanation.
Differences in the health status of the subjects in the present study
(healthy) and the previous study (patients) might be relevant to the
differences between the results, perhaps because the differences in
health status affected the preferential absorption route of TU (see
below).
Although no definitive explanation could be given for the
above-mentioned apparently conflicting results obtained by RIA and
GC-MS techniques, the changes in nonconjugated testosterone and TG in
plasma in the present study do agree with the results obtained in a
controlled study by Dehennin and Pérès (21), who
used a GC-MS analytical technique to monitor seven healthy subjects who
received a single oral combined dose of 40 mg of TU plus 1.5 mg of
epitestosterone undecanoate. Moreover, a sharp increase of TG
compared with nonconjugated testosterone in the plasma of one healthy
subject after a single oral dose of 80 mg of TU was observed by
Giagulli et al. (22), using RIA assay after paper
chromatographic purification.
It is generally considered that esterification of testosterone with
undecanoic acid shifts the route of absorption from the portal vein to
the lymph system (23) and that TU enters the blood via the
thoracic duct. However, the remarkable increase of TG in plasma after
oral TU administration observed here suggests that a substantial
percentage of TU may be absorbed via the portal vein, probably losing
the undecanoate group in the intestine, whereas the remaining
nonmetabolized TU could be absorbed via the lymphatic system, as
suggested by Coert et al. (24) in a controlled study of rats
after oral TU administration. This hypothesis is supported by the
observation that plasma TG was sharply increased post administration,
with the mean peak concentration being reached at 1 h after the
dosing, whereas unchanged TU was found in plasma of all the six
subjects receiving oral TU, with the mean peak concentration appearing
at 3 h after the dosing (14).
AG and EtG were increased in blood of all six subjects studied either
after oral administration of TU or after intramuscular administration
of combined TP plus TE. Thus, increases of AG and EtG in blood,
compared with their basal concentrations, seem to be indicative of
exogenous testosterone intake, regardless of the administration route.
Given the relevance of glucuronidation of testosterone, androsterone,
and etiocholanolone, analysis with and without hydrolysis of blood and
plasma samples appears necessary for obtaining the proper steroid
profile after exogenous testosterone intake, especially after oral
administration.
However, one subject showed no increase in TG but a very short increase
in nonconjugated testosterone, and longer-lasting clear increases of AG
and EtG after oral intake of TU. This subject did show a similar total
recovery of testosterone excreted in urine after TU administration
compared with the other five subjects (data not shown), as calculated
by the addition of its main urinary metabolites, thus ruling out the
possibility of poor gastrointestinal absorption of TU. Some other
possibilities for his abnormal kinetic results could be a faster
metabolism of testosterone to androsterone and etiocholanolone, a
specific enzyme deficiency for testosterone glucuronidation in
splanchnic organs, or again, a different balance between the portal
vein vs lymphatic absorption, as mentioned above.
At present, blood sampling is not a common practice in sports drug
testing, although expectations for its future use are strong,
especially for detecting the administration of peptide hormones
(25). In the present study, the steroid profile
(testosterone, TG, AG, and EtG) measured in blood spots dried on filter
paper correlated well with that obtained from plasma, with the
advantages of slightly higher concentrations being found in dried whole
blood, which suggest that blood cells may retain small portions of
testosterone and its glucuronide-conjugated metabolites. Thus, dried
blood spots on filter paper could be an optimal alternative to plasma
for assessing individual steroid profiles, with the advantages of the
small volume of blood needed, easy collection by fingerprick or
earprick, and less invasiveness. Blood TG/T obtained from dried blood
spots was increased until 8 h after administration, whereas other
plasma markers, TG/17OHP and TG/LH, were significantly increased
until 10 and 6 h, respectively, all confirming the signs of
administration of testosterone. It has also been verified in the
present study that steroids in dried blood spots are quite stable and
that the samples can be stored easily and transported from the field to
laboratories. Additionally, blood spots may be subjected to individual
DNA fingerprinting, which may offer definitive proof of the
authenticity of the sample. Taking into account the above-mentioned
merits of blood spots dried on filter paper as a sampling technique, it
could be feasible that a database of basal blood steroid concentrations
for each athlete be established and filed as historical
"fingerprints" for doping control. Thus, changes in blood
concentrations of TG, AG, and EtG, compared with historical basal
concentrations for each particular athlete, would be easily detectable
in dried blood spots. The existence of intact testosterone esters,
which also can be detected in blood spots dried on filter paper, may
offer additional unequivocal confirmation of testosterone intake. In
fact, traces of the unchanged testosterone ester of orally
administrated TU have been detected in some of these dried blood-spot
samples, as reported previously (14).
In conclusion, the ability of dried blood spots to provide reliable
information on the glucuronidation of testosterone and its main
metabolites (androsterone and etiocholanolone), which can be obtained
by analyzing only one or two dried blood spots on filter paper, make
this sample material a good alternative to plasma as a complement to
urinalysis for detecting testosterone abuse. However, it should
be kept in mind that the present proposed markers are derived from a
small group of subjects. For the wide application of blood TG/T, AG,
and EtG to detect testosterone abuse in doping tests, relevant
population reference values and intraindividual variations should be
taken into consideration and be studied carefully. The usefulness of
easily obtained capillary blood (from a fingerprick or earprick),
compared with drops of venous blood (studied in the present work),
should also be verified for widespread applicability of the proposed
approach.
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Acknowledgments
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We thank the Spanish and Catalonian research administrations
(projects FIS 96/1050, CICYT SAF 97-068, and CIRIT DOGC 2320) for
financial support. S-H. Peng also thanks the Spanish Ministry of
Science and Education for a postdoctoral fellowship. We thank Pere
Roset, Marta Mas, and Esther Menoyo for their work in the clinical
trials and some assistance in statistical analysis.
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Footnotes
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1 Nonstandard abbreviations: TG, testosterone glucuronide; EG, epitestosterone glucuronide; TU, testosterone undecanoate; AG, androsterone glucuronide; EtG, etiocholanolone glucuronide; 17OHP, 17-hydroxyprogesterone; T-D3, testosterone-[16,16,17]-d3; Et-D4, etiocholanolone-[2,2,4,4]-d4; TP, testosterone propionate; TE, testosterone enanthate; GC, gas chromatography; MS, mass spectrometry; TMS, trimethylsilyl; LH, luteinizing hormone; and TG/T, testosterone glucuronide/testosterone. 
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Abstract
Introduction
, 1 day before administration (-1 day);