APPROACHES TO THE DETECTION OF
STEROID ABUSE IN VETERINARY SPECIES
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APPROACHES TO THE DETECTION OF
STEROID ABUSE IN VETERINARY SPECIES
James P. Scarth
(Ghent University, Belgium and HFL Sport Science, UK)
Thesis for submission in fulfilment of the requirements
for the degree of Doctor (Ph.D) in Veterinary Sciences
Promoter: Prof. Dr. H. De Brabander (Ghent University, Belgium)
Co-promoter: Dr. J. Kay (University of Strathclyde, UK)
Co-promoter: Dr. L. Vanhaecke (Ghent University, Belgium)
Rector: Prof. Dr. P. Van Cauwenberge (Ghent University, Belgium)
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TABLE OF CONTENTS
PART 1: DETECTION OF ‘ENDOGENOUS’ STEROID ABUSE IN
Chapter 1: Presence, metabolism
‘endogenous’ steroid hormones in food producing animals
Chapter 2: Validation and application of an analytical biomarker
approach for the detection of nandrolone abuse in the porcine
Chapter 3: Validation of analytical biomarker approaches for the
detection of androgen, oestrogen and progestagen abuse in the
PART 2: DETECTION OF ‘DESIGNER’ STEROID ABUSE IN
Chapter 4: Steroid metabolism and detection in the equine
Chapter 5: Assessment of the applicability of in vitro
technologies to study drug metabolism in the equine
Chapter 6: Metabolism of the ‘designer’ steroid estra-4,9-diene-
3,17-dione in the equine and comparison to human and canine
CHAPTER 7: GENERAL DISCUSSION
and detection of
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atmospheric pressure ionization.
British Horseracing Authority.
a steroid based on the estrane nucleus.
a steroid based on the androstane nucleus.
a steroid based on the pregnane nucleus.
a steroid based on the cholane nucleus.
a steroid based on the cholestane nucleus.
collision activated dissociation.
European Community Reference Laboratory.
enzyme linked immunosorbent assay
enhanced product ion scan.
endogenous reference compound.
Food and Drug Administration.
Federation Equestre Internationale.
full width at half maximum height.
gas chromatography combustion isotope ratio mass spectrometry.
gas chromatography-mass spectrometry.
gas chromatography-tandem mass spectrometry.
Greyhound Board of Great Britain.
higher-energy collision decomposition.
high performance liquid chromatography.
high resolution-liquid chromatography-mass spectrometry.
International Conference of Racing Analysts and Veterinarians.
International Federation of Horseracing Authorities.
International Laboratory Accreditation Cooperation.
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MSTFA – N-Methyl-n
MTBSTFA – N
International Union of Pure and Applied Chemistry.
liquid chromatography-mass spectrometry.
liquid chromatography-tandem mass spectrometry.
lower limit of quantification.
limit of detection.
limit of quantification.
linear trap quadrupole.
minimum required performance limit.
mass to charge ratio
nicotinamide adenine dinucleotide.
nicotinamide adenine dinucleotide phosphate.
nuclear magnetic resonance.
national monitoring programme.
programmable temperature vaporiser.
quantitative real time reverse transcriptase polymerases
chain reaction technology
relative standard deviation (also known as coefficient of
Scientific Committee on Veterinary Measures relating to
selected reaction monitoring.
surface plasmon resonance.
thin layer chromatography-fluorescence.
time of flight.
atomic mass unit
upper limit of quantification.
World Anti-Doping Agency.
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Steroid structure and pharmacology
If one were to ask a member of the public what images the word ‘steroid’ conjured into
their imagination, the use of anabolic-androgenic steroids (AASs) in athletics or
bodybuilding would no doubt rank near the top of the list. However, steroids have a
range of structures and pharmacological actions that reach far beyond the anabolic
effects of AASs. The term ‘steroid’ itself refers to any compound possessing the basic
perhydrocyclopentanophenanthrene nucleus (Figure 1) (Makin, 1995).
Figure 1 – A) the perhydrocyclopentanophenanthrene nucleus, on which all steroids are
based and B) cholesterol as an example. Each carbon is assigned a number and the
four hydrocarbon rings are numbered A-D, as shown.
The nomenclature of this class of compounds is complex and large arrays of different
systems are used. These include; the official International Union of Pure and Applied
Chemistry (IUPAC) recommended systematic nomenclature (IUPAC, 2010), a range of
‘trivial’ or ‘common’ names and those of some proprietary preparations. Additionally,
many organisations use their own nomenclature (for example the company Steraloids).
However, these often deviate from the IUPAC recommendations. The choice of how to
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best name a steroid in a particular situation is, therefore, dependent on a number of
factors. If one were to always use only the IUPAC systematic name, then this could
make the text difficult to read for a non-expert. However, inappropriate over-use of trivial
names does not always give enough information in order to inform the reader. Therefore,
a combination of systematic and trivial names is often employed as a pragmatic
compromise (such as described by Makin et al. 1995) and this will be used in the current
text. As an example of the different ways of naming a steroid, some options for
testosterone are given below:
Trivial name: testosterone.
IUPAC systematic name: 17β-hydroxy-androst-4-en-3-one.
Proprietary example (containing testosterone esters): Sustanon.
When depicted in the orientation shown in Figure 1, substituents on the steroid backbone
may protrude below or above the plane of the paper and are drawn as such using either
a dashed or solid wedge respectively (indicating the stereochemistry α and β
respectively). Hydrogens in positions 8, 9, 10, 13 and 14 (when present) take β, α, β, β,
and α orientation respectively in all steroids discussed in this manuscript so their
stereochemistry will not be shown in any of the subsequent diagrams. A substituent in
position 5 may take either the α or β form, so hydrogens in this position will always be
labelled. A wavy line indicates that stereochemistry is unspecified.
In order to aid in the systematic naming of steroids, a number of different hydrocarbon
backbones are specified for use by IUPAC. These differ in the number and orientation of
carbons, which range from the 17-carbon (C17) gonane nucleus to the 27-carbon (C27)
cholestane nucleus (on which cholesterol is based). The range of steroid backbones
used in systematic nomenclature is shown in Figure 2. In this text, when describing the
trivial name for the oestrogens, the English version will be used (as opposed to the USA
use of estrogens). However, when systematically naming steroids that are based on the
estrane nucleus, the ‘o’ will not be used (in accordance with IUPAC guidelines).
No endogenous and very few exogenous steroids are based on the gonane nucleus. The
oestrogens and nandrolone (17β-hydroxy-estr-4-en-3-one) are based on the estrane
nucleus. The majority of androgens are based on the androstane nucleus and the
majority of progestagens and corticosteroids are based on the pregnane nucleus. Most
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of the bile acids are based on the cholane nucleus and sterols such as cholesterol are
based on the cholestane nucleus.
Gonane (C17) Estrane (C18)
Androstane (C19) Pregnane (C21)
Cholane (C24) Cholestane (C27)
Figure 2 – the range of hydrocarbon backbones used in steroid nomenclature.
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While many steroids are known to be endogenous (discussed further in chapter 1), a
wide range of exogenous steroid structures have been synthesized by chemists over the
years in order to optimise their biological properties. Pharmacologically, steroids possess
a range of activities far more diverse than their seemingly similar structures may
suggest. The following discussion considers the major effects of different steroid classes
in mammals. There are some subtle differences between various species, but these will
not be considered here since it is only a general overview.
Cholesterol (cholest-5-en-3β-ol – Figure 1) is derived from dietary intake, but is also
synthesized in the body. Cholesterol acts to regulate the fluidity of cell membranes and is
the precursor to the endogenous androgens, oestrogens, progestagens, corticosteroids,
vitamin D, the bile acids and, in certain species, to pheromones such as the 16-
androstenes (Hadley and Levine, 2006).
Bile acids such as cholic acid (3α,7α,12α-trihydroxy-5β-cholan-24-oic acid – Figure 3)
are secreted by the gall bladder into the intestine where they aid the absorption of lipids
into the body by reducing their surface tension (Hadley and Levine, 2006).
Figure 3 – cholic acid (a bile acid).
Progestagens such as progesterone (pregn-4-ene-3,20-dione – Figure 4) are produced
by both males and females in the adrenal glands and gonads. However, they are
secreted in much higher concentrations by females during certain stages of the ovulatory
cycle (including by the corpus luteum) and during pregnancy (Hadley and Levine, 2006).
Progestagens produce the majority of their effects through agonism of the progesterone
receptor. This leads to an increased metabolic rate, changes in breast morphology and
development/maintenance of the uterus/oviduct before and during pregnancy (Hadley
and Levine, 2006). Progestagens (and synthetic progestins) may also be used as
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contraceptives in females, which act by suppressing endogenous gonadotrophin release
and by inhibiting sperm penetration due to a change in viscosity of the cervical mucous
(Westhoff et al. 2010).
Figure 4 – progesterone (a progestagen).
Corticosteroids are produced by the adrenal cortex and fall into one of two broad
classes, depending on their predominant mechanism of action. However, there is some
overlap in the effects of the two classes. Glucocorticoids such as cortisol (11β,17α,21-
trihydroxy-pregn-4-en-3,20-dione – Figure 5a) agonise the glucocorticoid receptor and
act to regulate inflammation and immunity as well as fat, protein and carbohydrate
metabolism (Hadley and Levine, 2006). Mineralocorticoids such as aldosterone (11β,21-
dihydroxy-3,20-dioxo-pregn-4-en-18-al – Figure 5b) agonise the mineralocorticoid
receptor and act to maintain sodium and potassium balance (Hadley and Levine, 2006).
Figure 5 – A) cortisol (a glucocorticoid), B) aldosterone (a mineralocorticoid).
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Androgens are produced by both males and females in the adrenal glands and gonads.
However, they are secreted in much higher concentrations by male gonads.
Testosterone (17β-hydroxy-androst-4-en-3-one – Figure 6) is the most abundant
circulating androgen in males, but requires reduction in position 5 to produce the fully
active androgen, 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one – Figure 6).
The androgenic effects of these steroids are produced by agonising the androgen
receptor (AR) (Hadley and Levine, 2006). Androgens produce both androgenic
(masculinising) and anabolic (growth promoting) effects to varying degrees. This leads to
their more correct classification as anabolic-androgenic steroids (AASs). The androgenic
effects are the characteristic male secondary sexual features such as facial/body hair
growth and deepening of the voice, while the anabolic effects are predominantly muscle
and bone growth (Hadley and Levine, 2006). Whether or not all the anabolic effects of
AASs are mediated through the AR is currently unknown. Another possible mechanism
of action is antagonism of the glucocorticoid receptor (subject discussed further in
Figure 6 – testosterone and its conversion to the more
active androgen 5α-dihydrotestosterone.
Oestrogens such as oestradiol (estra-1,3,5(10)-triene-3,17β-diol – Figure 7) are
produced by both males and females in the adrenal glands, gonads and adipose tissue.
However, they are secreted in much higher concentrations by females during certain
stages of the ovulatory cycle (including by the corpus luteum) and during pregnancy
(Hadley and Levine, 2006). Oestrogens produce the majority of their effects through
agonism of the oestrogen receptor. This leads to breast growth and redistribution of fat
within the body, development/maintenance of the uterus before and during pregnancy,
changes to skin morphology and they are also important for bone growth (Hadley and
Levine, 2006). Oestrogens are also used as female contraceptives, which act primarily
by suppressing endogenous gonadotrophin release (Westhoff et al. 2010).
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Figure 7 – oestradiol (an oestrogen).
A wide range of synthetic AASs, oestrogens, progestagens and corticosteroids have
been produced over the years with the aim of enhancing their pharmaceutical qualities.
Modifications that have been applied to the majority of the steroid classes include
alkylation (in order to produce more orally active versions), esterification (to prolong
duration of action), acetylation (to enhance absorption) and halogenation (to enhance
potency) (Kicman, 2008). Specifically relating to AASs, the addition of a double bond at
position 1, the attachment of a pyrazole group to the A-ring or the removal of the 19
methyl group have been employed in order to increase the anabolic to androgenic ratio
and/or to inhibit their conversion to oestrogens (Kicman, 2008). Similar modifications to
glucocorticoids have been engineered in order to try and maximise the glucocorticoid to
mineralocorticoid effect ratio. Figure 8 shows a range of different synthetic AASs.
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Figure 8 – structures of the synthetic AASs methyltestosterone (17β-hydroxy,17α-
dien-3-one), mesterolone (17β-hydroxy,1α-methyl,5α-androstan-3-one), stanozolol (17β-
Steroid use in food production and competitive sports
The above discussion highlighted the potential anabolic effects of AASs in mammals. In
some species, however, oestrogens and progestagens may also produce anabolic
effects. In addition to effects on muscle and bone, steroids may also affect the pattern of
fat deposition within the body, leading to differential partitioning of muscle and fat;
although this depends on the steroid, species and sex of animal in question (Heitzman,
1975, Lone, 1997). Corticosteroids may produce some positive metabolic effects
following initial administration, but long-term use of high doses produces a general state
of catabolism within the body (Hadley and Levine, 2006).
Because of their potential anabolic effects, some steroids have been used to boost the
mass and quality of animal carcasses in food production for economic reasons