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Articles |
-dihydrotestosterone heptanoate: changes in urinary hormone profile
1
Drug Control Centre, King's College London, Manresa Rd., London SW3 6LX, UK.
2
School of Life Sciences, Kingston University, Kingston
upon Thames, Surrey KT1 2EE, UK.
a Author for correspondence. Fax 44-171-351-2591; e-mail a.kicman{at}kcl ac.uk.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone (DHT) doping in male athletes proposed the use
of the urinary concentration ratio of DHT to epitestosterone (EpiT) as
the primary marker and those of 5-androstane-3,17ß-diol
(5-Adiol) to EpiT, luteinizing hormone (LH), and
5ß-androstane-3,17ß-diol (5ß-Adiol) as secondary markers. Here
we investigate the effects on these markers of intramuscular
administration of DHT heptanoate (250 mg) to six healthy men. Within
24 h of administration all four markers greatly exceeded the
published discrimination limits, remaining above these limits for
10–14 days. All ratios returned to basal values by day 28. In contrast
to results after percutaneous administration, 5ß-Adiol excretion
decreased, probably as a consequence of greater suppression of
testicular steroidogenesis. Results were largely in agreement with
those obtained after percutaneous administration, although probably
augmented by the larger dose and the different route of delivery. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reduced metabolite of T), which is known not to perturb the T/EpiT
ratio (2). Recently, we proposed a confirmatory procedure
for detecting 5-DHT in male athletes (3). The urinary
concentration ratio of DHT/EpiT was chosen to be the primary marker for
detection of DHT doping; 5-androstane-3,17ß-diol (5-Adiol;
the main metabolite of DHT)/EpiT, 5-Adiol/luteinizing hormone (LH),
and 5-Adiol/5ß-androstane-3,17ß-diol (5ß-Adiol; a
metabolite of T) were chosen as secondary markers.
Because we developed a method capable of detecting percutaneous DHT
administration (3), we wanted to investigate the
robustness of the test by studying how well these markers were suited
to the detection of DHT doping when other routes of administration were
used. Oral administration may be convenient for the cheater, but for
steroids that are produced endogenously, it is not the ideal route of
delivery because of extensive first-pass metabolism. Nevertheless, DHT
may be taken orally by some sports competitors, and some work has been
done in this area (4)(5). First-pass
metabolism can be circumvented by administering formulations designed
for sublingual absorption, but nonetheless some of the dose may be
swallowed. Investigations into the changes in the urinary steroid
profile after sublingual application to four male volunteers
(6)(7) concluded that DHT/E,
5-Adiol/5ß-Adiol, and androsterone/etiocholanolone (A/E) were
suitable parameters of detection.
Intramuscular administration and, in particular, injection of esterified compounds prolong the activity of steroids because of a depot effect. Investigations of the possible clinical use of crystalline DHT (8) and DHT heptanoate (9) found that intramuscular injection gave a prompt and sustained increase in DHT, and concluded that such preparations could provide an effective and convenient method of replacement therapy. Such a future clinical use would increase the risk of underground availability of licensed DHT compounds. Even without such a supply, esters of DHT would be relatively simple to synthesize by the underground chemist and could easily be formulated for intramuscular delivery.
In a previous pilot study (2) to detect DHT abuse in the
field of sports, two male volunteers were injected intramuscularly with
DHT heptanoate, and the subsequent perturbations in the urinary hormone
ratios were evaluated with the use of peak height abundances, as
determined by GC-MS. Our primary objective in this study was to
quantify by GC-MS the changes in urinary steroid concentrations and
hormone concentration ratios after intramuscular administration of DHT
heptanoate to six men. We formulated a dose of 250 mg, equivalent in
mass to licensed formulations of T heptanoate, e.g.,
Primoteston®. For synthesis of the heptanoate ester,
5-DHT was reacted with heptanoyl chloride rather than heptanoic
anhydride to eliminate the possibility of heptanoic acid being
generated in the reaction. For quantification of urinary hormones a
mixture of internal standards was used, as described previously
(3), although a trideuterated analog of 5-DHT
(D3-DHT), synthesized by hydrogenation of D3-T
as described herein, was also included.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis of dht heptanoate
Synthesis of DHT heptanoate was done by acylation of the
17ß-hydroxyl group of DHT. DHT (25 g) was dissolved in
dichloromethane (250 mL). Heptanoyl chloride (16 mL) was added,
together with 4-(N,N-dimethylamino)pyridine (3.75
g) as a catalyst. The flask was stoppered with an anhydrous calcium
chloride tube, the entire apparatus was protected from the light, and
the reaction mixture was stirred magnetically at regular intervals.
After 1 week the reaction mixture was washed with sodium hydroxide
(2 x 75 mL, 1 mol/L), hydrochloric acid (2 x 75 mL, 1
mol/L), and then deionized water until an aqueous layer of neutral pH
was obtained. Evaporation of the organic layer resulted in a yellow,
waxy solid that was further dried in a desiccator for several days. The
product was recrystallized with the use of acetone/water and
characterized by electron impact full-scan MS with an ion-trap detector
(ITD 800; Finnigan MAT) coupled to a gas chromatograph (Model 5890A;
Hewlett-Packard) fitted with a HP-1 methyl silicone capillary column
(3). Purity of the compound was also assessed by nuclear
(1H) magnetic resonance spectroscopy (AMX 400 NMR
spectrometer; Bruker Spectrospin). The DHT heptanoate was prepared for
injection (250 mg in 1 mL of arachis oil and benzoyl alcohol solution)
at St. Thomas' Hospital, London.
synthesis of [16,16,17-2h3]5-dht
[16,16,17-2H3]T (20 mg) was
dissolved in tetrahydrofuran (10 mL), a solvent reported to favor the
production of the 5- over 5ß-isomer during hydrogenation reactions
involving 3-oxo-4-ene compounds and without any of the starting
material remaining (10). A catalyst (palladium on
activated charcoal, 20 mg) was added, and the mixture was hydrogenated
for 2 h while being stirred. The reaction products, the 5- and
5ß-isomers of [16,16,17-2H3]DHT, were
separated by means of their differing solubilities in
acetonitrile/H2O. The product containing both isomers was
dissolved in the minimum volume (450 µL) of acetonitrile, but
addition of an approximately equal volume of water, to our surprise,
caused precipitation of a portion of the 5-isomer. This portion was
isolated by removal of the supernatant after centrifugation. The
chemical and isotopic purity of this
[16,16,17-2H3]5-DHT was assessed by
full-scan MS. With the use of a 25-m HP-1 methyl silicone column
(Hewlett-Packard) and operating conditions described elsewhere
(3), the retention time/methylene units of the
bis-trimethylsilyl derivatives of D3-5- and
D3-5ß-DHT (m/z 437) were 22.2 min/26.21
methylene units and 17.0 min/24.56 methylene units, respectively.
administration and sample collection
5-DHT heptanoate (250 mg) was administered intramuscularly to
six healthy male volunteers (ages 23 to 28 years). Ethical permission
and informed consent were obtained in accordance with our institution.
Each subject gave a brief medical history and stated that they were not
taking any medication likely to interfere in the study nor were they
competing athletes at county level or above. Urine samples (24-h
pooled) were collected on days -2 to 5 and on days 7, 10, 14, 21, and
28, except on the day of administration, when the collections were
divided into two 12-h periods. Total volumes were recorded, and the
samples were divided into appropriate aliquots. Urine samples for
steroid analysis were stored at -20 °C, and for LH analysis samples
were frozen rapidly in liquid nitrogen and then stored at -70 °C.
analysis of urine
Urinary steroid concentrations and the A/E peak-height ratio were
determined by selected ion monitoring GC-MS as described elsewhere
(3). The trideuterated internal standard mixture consisted
of D3-DHT in addition to D3-T,
D3-EpiT, and D3-5-Adiol, in final
concentrations of 50, 50, 50, and 100 µg/L, respectively.
Urinary LH concentration was determined with the Serono immunoenzymetric assay, both by direct measurement and after ultrafiltration, according to the method described previously (3).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and 5ß-isomers of D3-DHT; separation of the two
isomers produced 4.19 mg of D3-5-DHT. Validation of the steroid assay has been described elsewhere (3). The four quality controls analyzed in each assay (n = 6 assays) showed a similar between-assay imprecision and gave urinary concentrations of steroid analytes within 2 SD of the mean values reported previously.
In our previous paper (3), the unpaired Student's
t-test rather than the paired t-test was used in
determining the statistical significance between samples collected
before and after DHT administration. We used the unpaired test because
in the context of sports the detection of doping with endogenous
steroids by comparing changes in an individual's urinary hormone
profile over time (longitudinal profiling) requires collection of
multiple samples and is therefore relatively expensive and
time-consuming. Although a similar statistical evaluation of
intramuscular administration data would be preferable, the wide range
of basal values observed in this study together with the considerable
variation in individual responses to intramuscular injection gave a
data set that was not thought to form part of a normal distribution.
For this reason, and also because of the limited number of
observations, nonparametric statistics were applied, and the
significance of changes in the urinary hormone profile was assessed by
a one-sided Mann–Whitney test. The threshold for significance was
chosen at P 
0.05. Mean excretion rates after DHT
heptanoate administration are shown in Fig. 1
, together with a profile of the maximum and minimum daily
excretions to give an indication of the spread of the data. After
injection, the mean 24-h excretion rates of DHT and 5-Adiol
increased significantly compared with the basal mean [day -1 and day
-2 shown not to differ significantly (two-sided Mann–Whitney test)],
maximizing within the first 24 h of sampling with the rates ~8
times basal. Excretion rates remained significantly augmented
(one-sided Mann–Whitney test; basal < administration) until day
10 and still did not return to basal concentrations until day 28. The
wide separation of the maximum and minimum excretion profiles indicates
the large degree of variability in individual responses, particularly
in the period immediately after administration. Despite this
variability, the excretion profiles of all individuals were found to
follow a similar trend.
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Accompanying the increase in DHT and 5-Adiol was a decrease in
excretion rates of T, EpiT, 5ß-Adiol, and LH (Fig. 1
). T, EpiT, and
LH all followed a similar pattern of decline, with maximum suppression
for all three analytes occurring by days 4 or 5 and approximating 20%
of the basal concentrations. Even with the wide range of basal values
shown in the graphs, excretion rates of all three analytes were
significantly suppressed (one-sided Mann–Whitney; basal >
administration) between days 1 and 7, with T showing additional
significance on day 10. With 5ß-Adiol, the fall in excretion rate was
not found to be significant because of the wide range of basal values.
However a decrease from basal was observed in all six volunteers, and
if excretion rates were first calculated as a percentage of the values
obtained on day -1, then administration values were found to be
significantly lower (one-sided Mann–Whitney; basal >
administration) than basal (day -2) between days 1 and 14.
From our previous study, the hormone ratio DHT/EpiT was proposed as the
primary marker, with 5-Adiol/EpiT, 5-Adiol/LH, and
5-Adiol/5ß-Adiol all as secondary markers with discrimination
limits of 2, 11.6, 112.4, and 4.3, respectively. Values exceeding these
limits were shown to be indicative of doping with DHT (3).
The responses to intramuscular administration can be seen in Fig. 2
, in which ratios of samples from each individual are displayed
at seven selected times: two at basal, one each at the period where the
applicable discrimination limit was first exceeded, at the maximum, at
the times where the ratio decreased to just above and below the limit,
and finally at 28 days after administration. For any individual, when a
ratio in all the samples collected did not exceed the corresponding
limit, then selected points were plotted to give an illustration of the
changes in ratio over time.
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After administration, ratios of all volunteers increased rapidly, in
most cases exceeding the given discrimination limits within the first
24 h after injection and remaining above these limits until about
day 10. All ratios had returned to basal by day 28. Although all
individuals responded in a similar way, there was considerable
variation in the degree of response. One volunteer in particular had
relatively low basal values and although he did respond to
administration, his ratios exceeded only the discrimination limit for
5-Adiol/5ß-Adiol. Nevertheless, statistical evaluation of the data
(one-sided Mann–Whitney; basal < administration) showed all
ratios for the group to be significantly augmented from day 0 to day 7.
The histograms in Fig. 2
show how many of the samples exceeded the
discrimination limits on each day. With the exception of the ratio of
5-Adiol/5ß-Adiol, five of the six volunteers gave samples for
which the ratios exceeded the discrimination limits. The one exception
was the samples collected from the individual with low basal
concentration ratios, which resulted, in part, from the urine having a
larger average basal EpiT excretion than the other volunteers (218
µg/24 h compared with a range for the rest of the group of 21–116
µg/24 h). With 5-Adiol/5ß-Adiol, two out of the six volunteers
gave samples whose ratios did not exceed the discrimination limit;
samples from these volunteers were characterized by having relatively
large average basal excretions of 5ß-Adiol compared with the rest of
the group (630 and 534 µg/24 h compared with a range for the rest of
the group of 55–160 µg/24 h).
Changes in the T/EpiT ratio after administration (Fig. 3
) were not significant (one-sided Mann–Whitney; basal <
administration) as a group. Nonetheless, in five of the six
individuals, the T/EpiT ratio in all samples collected during the first
3 days after administration was greater than the respective basal
ratio.
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The peak-height ratio of A/E was also determined on selected days (Fig. 4
), this being a marker chosen by some IOC-accredited
laboratories. The A/E ratio was found to be significantly increased
(one-sided Mann–Whitney; basal < administration) between days 1
and 5, increasing ~3-fold in the first 24–48 h, and then slowly
returning to basal.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reduced metabolite
5-Adiol, while decreasing the excretion rates of the hormones T,
EpiT, and LH. Similar findings occurred with intramuscular
administration, although in general the responses were more marked, a
fact we attribute to the larger dose and different route of
administration. With an intramuscular route of delivery, all the drug
injected is bioavailable, whereas with percutaneous administration,
only ~10% of the dose is able to penetrate the skin. Excretion rates
of DHT and 5-Adiol rose ~8-fold, maximum excretion being attained
within the first 24 h after administration and then slowly
declining. T, EpiT, and LH also showed a more marked response with
excretion rates decreasing to ~20–25%. This pattern of suppression
was consistent with the administered DHT causing a negative feedback
effect on the hypothalamic–pituitary–testicular (HPT) axis.
The hormone concentration ratios DHT/EpiT, 5-Adiol/EpiT,
5-Adiol/LH, and 5-Adiol/5ß-Adiol all increased after
percutaneous administration, and with the exception of
5-Adiol/5ß-Adiol, mean increases for the group exceeded the
discrimination limits. With intramuscular administration the increases
in concentration ratios were more marked, the means for the group
clearly exceeding the discrimination limits of all four markers.
Consistent with a depot preparation, these effects were sustained, and
by day 10 the ratios still exceeded the respective limits in all cases
except 5-Adiol/5ß-Adiol. A return to basal values did not occur
until about day 28.
On an individual basis, five of the six volunteers administered
intramuscular DHT had concentration ratios that for several days
exceeded the discrimination limits for DHT/EpiT and 5-Adiol to EpiT
and LH. This compares with respective numbers of 6, 7, and 5 volunteers
out of the 10 from the percutaneous study, whose ratios exceeded the
discrimination limits on day 3 of administration, the day in which
ratios for the group were most augmented. Therefore, under our proposed
confirmatory procedure, five of the six volunteers intramuscularly
administered DHT heptanoate would be considered positive. That one
volunteer would have escaped detection, despite the dose administered
and its direct route of delivery, might suggest that our discrimination
limits are too large. This favors investigating the development of a
discrimination function incorporating several hormone concentration
ratios.
The implementation of longitudinal profiling in all IOC laboratories would also be particularly useful for individuals whose concentration ratios did not exceed the discrimination limits but were nevertheless significantly increased above basal values. By comparison of the urinary hormone profiles of individuals over a period of time, a more sensitive test based on differences in ratio could be established, and thus detection of doping would depend on the significance of changes in concentration ratios as opposed to the ratios themselves exceeding discrimination limits.
A decrease in the excretion rate of 5ß-Adiol was observed in all six individuals, although the overall change was not found to be significant because of the considerable range of basal values observed. However, when individual changes in excretion rate were analyzed as a percentage of the basal value for that volunteer, decreases in the excretion rate of 5ß-Adiol were found to be significant. This result contrasts with the findings of the percutaneous study, where there appeared to be no change in the individual rates of 5ß-Adiol excretion despite suppression of the HPT axis. 5ß-Adiol glucuronide (G) is believed to have more than one origin (3). After DHT administration, 5ß-Adiol from an extrasplanchnic source would be expected to fall, suppression of the HPT axis resulting in a decrease in availability of one of its major precursors, T. 5ß-Adiol G arising from hepatic metabolism of adrenal precursors would however be expected to remain unaffected. Such a situation would result in an overall decrease in 5ß-Adiol G excretion rates, and this appears to be the case with intramuscular administration. With percutaneous administration there was also suppression of the HPT axis, and although this was not so marked, we expected to see some change in 5ß-Adiol G excretion. That the excretion rates remained unchanged is difficult to explain, but it is possible that the adrenal contribution to the pool of urinary 5ß-Adiol G is perhaps the more important, the decrease from an extrasplanchnic source having a lesser influence on the overall urinary excretion rate.
The decreases in the excretion rate of 5ß-Adiol G after intramuscular
administration, together with the larger increases in the excretion
rate of 5-Adiol G, are responsible for the greater response in the
ratio of 5-Adiol/5ß-Adiol compared with that observed with
percutaneous administration. Samples from 4 of the 6 volunteers had
ratios that exceeded the discrimination limit for 6 days after
injection, compared with only 2 of the 10 from the percutaneous study
that were greater than the limit on the day in which this ratio was
most augmented.
The peak-height ratio of A/E was not shown to be a sensitive marker of percutaneous DHT administration because steroids of adrenal origin contribute largely to the formation of G conjugates of A and E. The A/E ratio, however, is reported to be a good marker of oral administration (4)(5), and we suggested previously that large doses and administration by other routes may also have a greater effect on this ratio. After sublingual DHT administration (25 mg) (6), an ~7-fold increase in the concentration ratio of A/E was reported, although this ratio was the least sensitive of the ratios studied and remained above basal for the shortest time interval after administration. Consideration should also be given to the possibility that some of the sublingual formulation may have been swallowed, resulting in first-pass metabolism of DHT to AG.
Intramuscular injection caused an ~3-fold increase that was found to
be significant. However, compared with the concentration ratio of
5-Adiol/5ß-Adiol (Fig. 4
), the relative increases in the
peak-height ratio of A/E compared with basal are smaller.
5-Adiol/5ß-Adiol is our least sensitive marker in terms of
exceeding the respective discrimination limit, but in terms of
perturbation, it does nevertheless increase immediately after
administration in all individuals. Hence we favor the ratio of
5-Adiol/5ß-Adiol as a marker for longitudinal profiling, although
natural intraindividual variation would have to be fully explored
before we would recommend this ratio for such profiling. The general
stability of steroid profiles for doping control purposes is currently
being explored (e.g., [11]).
The concentration ratio of T/EpiT was expected to remain unchanged after DHT treatment because of suppression of the HPT axis, resulting in an equal reduction in the secretion of T and EpiT from the testis. The excretion rates of T did not, in fact, decrease as rapidly as those of EpiT, thus giving rise to a small increase in T/EpiT shortly after administration. Such changes were not significant as a group because of the spread of data and the limited number of individuals, but samples collected from five of the six individuals showed ratios that had approximately doubled over basal by 3 days after administration. This result could possibly be explained by the greater binding affinity of DHT compared with T with plasma sex hormone-binding globulin. DHT generated from the administration of the heptanoate ester will displace T from sex-hormone-binding globulin, whereas EpiT, which is not bound by this protein, remains unaffected. Work by Pugeat et al. (12), while showing no change in the binding affinity of T after chronic percutaneous DHT treatment, did find an ~3-fold increase in the percentage of unbound T after acute percutaneous DHT administration (three doses of 250 mg). Given a similar situation after intramuscular injection, the increased amount of free T produced by displacement could partially compensate for the decrease in secretion of T from the testis.
In summary, the proposed confirmatory procedure for detecting DHT
doping in male athletes with the use of the concentration ratios DHT/E,
5-Adiol/E, 5-Adiol/LH, and 5-Adiol/5ß-Adiol was applied to
samples from volunteers administered intramuscular DHT heptanoate. The
discrimination limits of all four markers were exceeded, the larger
dose and the different route of administration augmenting the responses
seen previously with percutaneous administration. The markers were able
to detect a normal replacement dose of DHT heptanoate, a dose that
would be thought modest in terms of doping in sport, up to 10 days
after administration. This provides confidence, therefore, that doping
with DHT esters by sports competitors, either obtaining such
preparations from illegal synthesis or underground availability of
future licensed preparations, can be detected, and thus further
demonstrates the suitability of these hormone concentration ratios as a
method of confirming DHT abuse.
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Acknowledgments |
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Footnotes |
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-dihydrotestosterone; EpiT, epitestosterone (17-hydroxyandrost-4-ene-3-one); 5-Adiol, 5-androstane-3,17ß-diol; 5ß-Adiol, 5ß-androstane-3,17ß-diol; LH, luteinizing hormone; T, testosterone; A, androsterone; E, etiocholanolone; D3, trideuterated; HPT, hypothalamic–pituitary–testicular; G, glucuronide. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone doping in male athletes. Clin Chem 1995;41:1617-1627.
-dihydrotestosterone. Donike M Geyer H Gotzman A Mareck-Engelke U eds. Recent advances in doping analysis (3): proceedings of the 13th Cologne workshop on dope analysis, 12–17 March 1995 1996:201-213 Sport und Buch Strauß Köln. .
-metabolites. J Sports Med Phys Fitness 1995;35:235-250.
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