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The role of prohormones, ‘classic’ and ‘designer’ steroids, clenbuterol, peptide hormones and newer molecules causing concern in dietary supplements is discussed. Apart from their potential adverse effects on athletes’ health, their non-achievement of increased strength and muscle size, trace quantities present in contaminated dietary supplements can lead to failed doping tests. The methodologies used for the identification and determination of prohibited substances in very low concentrations, mainly liquid chromatography and mass spectrometry, are also addressed. Of concern is the anticipation that the number of dietary supplements containing (not yet) prohibited designer steroids and other performance-enhancing newer chemical entities will increase. Athletes, coaches and sports doctors should therefore be provided with information regarding dietary supplements and be advised to minimise risks for non-intentional ingestion of forbidden substances by using safe products listed on databases, such as those obtainable in The Netherlands and Germany.
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SAJS M VOL. 26 NO. 3 2014 87
REVIEW
e role of prohormones, ‘classic’ and ‘designer’ steroids, clenbuterol, peptide hormones and newer molecules causing concern in dietary
supplements is discussed. Apart from their potential adverse eects on athletes’ health, their non-achievement of increased strength and
muscle size, trace quantities present in contaminated dietary supplements can lead to failed doping tests. e methodologies used for the
identication and determination of prohibited substances in very low concentrations, mainly liquid chromatography and mass spectrometry,
are also addressed. Of concern is the anticipation that the number of dietary supplements containing (not yet) prohibited designer steroids
and other performance-enhancing newer chemical entities will increase. Athletes, coaches and sports doctors should therefore be provided
with information regarding dietary supplements and be advised to minimise risks for non-intentional ingestion of forbidden substances by
using safe products listed on databases, such as those obtainable in e Netherlands and Germany.
S Afr J SM 2014;26(3):87-90. DOI:10.7196/SAJSM.553
Dietary supplements containing prohibited substances:
A review (Part 2)
P van der Bijl, BSc Hons (Chem), BSc Hons (Pharmacol), BChD, PhD, DSc
Emeritus Professor and Former Head, Department of Pharmacology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, Cape Town,
South Africa; and Invited Foreign Professor, Department of Pharmacology, Pirigov’s Russian National Research Medical University, Moscow, Russia
Corresponding author: P van der Bijl (pietervanderbijlcpt@gmail.com)
In Part 1[1] the focus was on the
presence of stimulants in dietary
supplements, while the current
article deals with ‘classic’ and
‘designer’ steroids, clenbuterol,
peptide hormones and newer molecules that
cause concern in sports nutrition due to their
accidental intake as contaminants.
Prohormones
It has been shown in a previous study that
~15% of dietary supplements comprising
mainly vitamins, minerals, proteins and
creatine contained undisclosed anabolic
androgenic steroids.[2] These steroids were
mainly prohormones, referring to androgenic
precursors that are enzymatically activated in
vivo to the anabolic steroid testosterone and
its derivatives. In the aforementioned study,
it was assumed that these prohormones
were probably the result of contamination
before or during manufacturing processes.
Nevertheless, the quantities of prohormone
detected in these supplements could
have resulted in infringements of doping
regulations. Prohormones are viewed by
many consumers as natural compounds
to promote strength and muscle mass,
change body composition and improve
general feelings of wellness with fewer
adverse effects than testosterone itself
or other synthetic androgenic steroids.[3]
Furthermore, prohormones are perceived and
commercially promoted as legal alternatives
to testosterone, with comparable anabolic
eects. However, prohormones are listed as
prohibited substances on the World Anti-
Doping Agency (WADA) list and are illegal
to sell or import in many countries.
Cholesterol is metabolised by multiple enzyme
systems into testosterone via a number of
androgenic intermediates (prohormones),
including dihydroepiandrosterone (DHEA)
(Fig.
1). e biosynthetic pathway via DHEA
leads to the production of androstenedione
(DIONE) and androstenediol (DIOL). ese
intermediates can also be converted to the
oestrogens, which may cause gynaecomastia
and hepatic dysfunction. To counteract
Cholesterol
Pregnenolone
17-α-hydroxypregnenolone
Dehydroepiandrosterone (DHEA)
Androstenediol (DIOL)
Progesterone
7-α-hydroxyprogesterone
Androstenedione (DIONE)
Testosterone
Aromatase
Oestrone
Oestriol
Oestradiol
Fig. 1. Outline of major pathways in the biosynthesis of steroid hormones.
88 SAJSM VOL. 26 NO. 3 2014
these adverse effects, some athletes use
prohormones out of the competition season
in cycles lasting 4
-
12 weeks, either alone
or in a stacking manner, i.e. taking multiple
compounds with different oestrogenic
potential simultaneously. Alternatively,
users may follow a pyramid mode, taking
the highest doses in midcycle. Additionally,
selective oestrogen receptor modulators or
aromatase inhibitors (Fig.
1) may be taken to
attenuate oestrogenic eects, and androgenic
herbal products taken to diminish the low
period between cycles.
[3,4] However, two well-
conducted clinical studies have not shown
DHEA, DIONE or DIOL to produce any
advantageous anabolic or ergogenic eects
at all, but have conrmed the risk of adverse
eects. In particular, in one of the studies,
LDL-cholesterol/HDL-cholesterol ratios were
increased by 11%, signicantly elevating the
risk for cardiovascular disease and reducing
luteinising hormone levels, which may
decrease testicular and adrenal testosterone
production.[3] Other prohormones, e.g.
those of the much-used/abused anabolic
steroid 19-nortestosterone (nandrolone),
have also appeared on the market.[5] One of
these prohormones, norandrostendione,
administered as a single dose, has been
shown to lead to detectable urinary quantities
of the main metabolite (19-norandrosterone)
of the parent steroid nandrolone for a period
>10 days.[5]
Classic anabolic steroids
Steroid structures have perhydrocyclopenta-
nophenanthrene nuclei comprising four
rings (A, B, C and D), as represented by the
structure of testosterone (Fig.
2). Loci where
modifications occur as well as examples
of typical reactions, resulting in structural
modifications to the steroid nucleus, are
also shown. Furthermore, steroids can be
classi fied into six groups according to the
number of carbon atoms, i.e. gonanes (C17),
estranes (C18) (e.g. oestradiol and oestrone),
andro stanes (C19) (e.g. testosterone and
andro stenedione), pregnanes (C21) (e.g.
pro gesterone and cortisol), cholanes (C24)
(e.g. cholic acid and desoxycholic acid) and
chole stanes (C27) (e.g. cholesterol). With
the exception of the cholanes, steroids
are precursors or natural hormones that,
depending on their in vivo functions, can
be divided into oestrogens, androgens,
glucocorticoids and mineralocorticoids. e
steroid category of compounds includes the
classic anabolic steroids, e.g. metandienone,
stanozol, boldenone, oxandrolone,
dehydro -
chloromethyl-testosterone, etc.,
which have
been found in high amounts
(>1 mg/g) in
certain dietary supplements and vitamin
preparations that are freely available on the
market. ese steroid ingredients were either
listed on package labels under some other
chemical/non-approved name or not disclosed
at all. Concentrations of these steroids in the
supplements were of such orders that even
within the limits of recommended supplement
intake, supratherapeutic doses of these
substances would be ingested. Many athletes
and other individuals in the population at large,
including women, adolescents and children,
regularly consume dietary supplements in
quantities beyond the safe daily recommended
doses, and therefore adverse eects in these
groups of users could be severe. In men,
acne, testicular atrophy, prostate enlargement,
decreased spermatogenesis, infertility, impo-
tence and changes in libido may occur.[6]
Furthermore, gynaecomastia, which may
require surgical intervention, can also occur;
some male users of steroids concurrently use
tamoxifen or other agents to prevent or treat
this condition. In women using steroids,
acne, potentially irreversible masculinisation,
clitoris enlargement, menstrual irregularities
and changes in libido may result. In both sexes,
psychiatric eects, e.g. aggression (‘roid’ rage),
psychoses, manic episodes, panic disorders,
depression, etc. have been documented.
Long-term steroid use has also been associ-
ated with dependency and a withdrawal
syndrome associated with suicidal thoughts,
an increased incidence of tumours and
premature mortality.[6] Of particular concern
are the eects of steroids on cholesterol and
lipid metabolism, hypercalcaemia, electrolyte
and fluid disturbances, hypertension and
thrombotic events, e.g. emboli, myocardial
infarction and cerebrovascular accidents,
the latter two conditions possibly resulting
from an increase in platelet aggregation and
erythrogenesis. In adolescents and children,
virilisation and premature closure of the
epiphyseal plates, which may result in stunted
growth, have been described. Most of the
classic anabolic steroids are methylated in the
17-position of the D-ring of the perhydro-
cyclopentanophenanthrene nucleus (Fig.
2), a
molecular feature that is associated with high
hepatoxicity and carcinogenicity.[5]
While the commercial sources of these
steroids on the world market are not always
known, it appears as if many of them are
sourced from Chinese bulk manufacturers
and are intentionally incorporated into dietary
supplements by unscrupulous companies.[5]
Designer steroids
ese steroid molecules were synthesised some
5 decades ago and evaluated in preclinical
studies for their anabolic and androgenic
effects.[7] They are not listed as components
of any currently available pharmaceuticals
for clinical use, are not on the WADA list of
prohibited substances and are manufactured
exclusively for the dietary supplement black
reduction
19
18
O
OH
reduction methylation
alkylation
esterication
oxidation
glucuronidation
or sulphation
Cyclopentanoperhydrophenanthrene structure
hydroxylation
1
2
345
A B
C D
10
11
9
8
7
6
12
13
14 15
16
17
Fig. 2. Some structural modications of the steroid nucleus, using the testosterone molecule as
an example.
SAJS M VOL. 26 NO. 3 2014 89
market. Examples of such agents, to name but a few, are prostanozol,
methasterone and andostatrienedione, and to date more than 40
such
designer steroid molecules have been detected in laboratories.[7] Little
is known regarding their pharmacological actions and safety proles in
humans. ey are either listed under some other chemical/non-approved
name or are not disclosed at all on dietary supplement labels. Should
metabolites of these designer steroids be detected in an athlete’s urine,
doping infringement charges would probably ensue.
Clenbuterol
Many athletes who claim that they suer from asthma or exercise-
induced asthma use metered-dose inhaled β2-agonists for alleviating
their symptoms of bronchoconstriction. While the inhalational β2-
agonists such as terbutaline, albuterol and salmeterol are permitted
for use by athletes by the WADA, oral and injectable forms of these
pharmaceuticals are not. However, some of these β2-agonists, e.g.
clenbuterol, are considered anabolic substances by some sports-
governing agencies. Hitherto, two cases have been described in which
dietary supplements contained therapeutic and supratherapeutic
doses of 30
µg/tablet and 2
mg/capsule of clenbuterol, respectively.[8]
In the supratherapeutic preparation, which contained 100
times the
therapeutic dose of clenbuterol, the presence of this β2-agonist was not
disclosed on the package label.
Peptide hormones
Over-the-counter dietary supplements are frequently promoted by
manufacturers as being able to increase human growth hormone
levels in the body. However, a review has shown that while human
growth hormone does increase lean body mass, it has no benecial
eect on strength or exercise capacity in trained athletes.[9] However,
use of human growth hormones has been associated with higher
rates of so-tissue oedema, arthralgias and carpal tunnel syndrome.
Furthermore, dietary supplements advertised as having anabolic, fat-
reducing and anticatabolic properties, and containing the prohibited
growth hormone-releasing peptide-2 (GHRP-2), were detected a few
years ago. e presence of such substances may lead to inadvertent
doping infringements. While GHRP-2 itself is not specically barred
by the WADA for use by athletes, it is a releasing factor that belongs
to a prohibited substance group on their list.[7]
Other newer molecules
A selective androgen receptor modulator and agonists of the peroxi some
proliferator-activated receptor δ, which produce anabolic eects and
enhance endurance, respectively, have been found on the black market.[7]
It has also come to the attention of the WADA that another sub-
stance for increasing endurance, GW501516, has been available for
some time on the black market, through the internet and elsewhere.
Anti-doping authorities have already seen its use by athletes, as
there have been a number of positive cases. is developmental drug
has not been approved for clinical use anywhere in the world and
has been withdrawn from further investigation by pharmaceutical
companies due to its serious toxicity prole.[10]
Identication and determination of prohibited substances
While a wide variety of analytical techniques have been used in the past
to detect prohibited drugs in biological uids, rapid improvements
in mass spectrometry (MS) have allowed accredited laboratories to
develop specic and comprehensive screening methods that are able
to detect amounts of drugs and/or their metabolites in quantities
as low as 1
mg/L in urine.[4] Over many years, gas chromatography
(GC) has proven itself in laboratories to be a useful technique for
separating and identifying individual components in mixtures of
chemical compounds on various chromatographic columns in terms
of their retention characteristics (relative to an internal standard
compound), as well as for quantifying them. A variety of detectors,
e.g. ame ionisation detectors, thermal conductivity detectors and
electron capture detectors have been used for this purpose. However,
for the successful application of this technique, it is a prerequisite that
for compounds to be separated, they are volatile, usually aer the
necessary derivatisation steps. Samples may also have to be hydrolysed
to release steroid metabolites from their glucuronic acid conjugates,
usually by means of the enzyme β2-glucuronidase, which may result
in the generation of related steroidal compounds or incomplete
deconjugation. For the purpose of volatilisation, the mixtures to
be analysed are oen trimethylsilylated (TMS) prior to injection
onto GC columns. e more volatile TMS ethers of the individual
components are then distributed between gaseous and solid phases,
separated and detected. By feeding gaseous euents from a GC into
an MS, further improvement in identication and quantitation of
compounds in mixtures can be expected. However, this technique of
GC/MS is not so eminently suitable for routine clinical analysis, but
has better applications as a screening tool of prohibited compounds in
dietary supplements or urinary metabolites because of the high labour
intensiveness, high cost and relatively poor sensitivity. The poor
sensitivity is of relevance when samples are analysed for compounds
which have low thermal stabilities, e.g. steroids such as trenbolone
and gestrinone.[4]
In recent years, another chromatographic tool, i.e. liquid chroma-
tography (LC) coupled to ultraviolet or diode-array detectors,
has become increasingly important in drug-assaying laboratories.
However, similar to GC, this method is not sensitive enough to detect
trace levels of compounds and is not well suited for the identication
of new substances with unknown chemical and physical properties.
Coupled with MS, it provides specificity, precision and high
sensitivity, allowing the detection of very low quantities of polar and
non-polar compounds, a requirement being that the methodology
be extensively validated. LC/MS has high throughput capabilities,
requires small sample volumes, minimal sample preparation and
thermal stability is usually not a factor. Furthermore, if required, a
number of components can be identied and quantied in a single
analysis within the dynamic calibration range of the instrumentation,
which can span four orders of magnitude. Additionally, because of
the absence of hydrolysis or derivatisation steps, LC/MS can be used
to measure concentration ratios for steroids between conjugated and
free forms, thereby lowering the risk of false positive or misleading
outcomes.
The use of ultra-high performance LC has further improved
chromatographic resolution, thereby lowering the possibility of
missing potentially important co-eluting analytes, and critical
pairs of isomers may be separated and detected. Coupled with high
acquisition rate mass analysers such as triple quadrupole MS in
tandem with LC/MS(/MS), exceptionally powerful techniques have
90 SAJSM VOL. 26 NO. 3 2014
evolved in the detection of steroids with marginal GC properties,
for example.
[11-
13] Methodologies such as these have enabled the
identification of characteristic product ions of common steroid
structures and nuclei.[11] ese powerful analytical tools provide a
means of detecting a wide variety of unknown steroids based on
common chemical structural properties, new metabolites, as well
as new designer steroids (likely to be added to dietary supplements)
made to circumvent anti-doping controls.
Conclusion
Dietary supplement use among athletes to enhance performance
is proliferating as more individuals strive to obtain a chemical
competitive edge. As a result, the concomitant use of dietary
supplements containing performance-enhancing substances that fall
in the categories outlined in the current review can also be expected
to rise. This is despite ever-increasing, sophisticated analytical
methodology techniques being used to assay dietary supplement
and urine samples in doping laboratories. The reasons for this
include: a variety of these chemical entities, many of them on the
prohibited drug list of the WADA, are being produced commercially
in factories around the world; aggressive marketing strategies are
being employed by companies; and these supplements can be easily
ordered, for example via the internet. It can also be anticipated that
there will be an increase in the number of supplements containing
designer steroids and other newer molecules. Chromatographic
techniques combined with MS, leading to identication of molecular
fragments and product ions, will assist in determining these
substances. To prevent accidental doping, information regarding
dietary supplements must be provided to athletes, coaches and sports
doctors at all levels of competition. In South Africa, this situation
is complicated by the fact that the system of Acts and Bills lacks
specicity regarding nutritional supplements, including the listing of
prohibited substances on package labels.[14,15] e risks of accidental
doping via dietary supplement ingestion can be minimised by using
safe products listed on databases, e.g. such as those available in e
Netherlands and Germany.[5] Finally, athletes must be reminded that
if they test positive for a prohibited substance not disclosed on the
package label of a supplement, it would constitute a doping violation,
with all the consequences thereof.
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... Esses compostos foram, de maneira geral, sintetizados nos anos 1960 e testados apenas em animais, com o objetivo de averiguar seus efeitos anabólicos e androgênicos. Essas substâncias não são produzidas por nenhuma empresa farmacêutica com o intuito de uso clínico, sendo sintetizadas exclusivamente pelo "mercado negro" de suplementos (Cavalcanti et al., 2013;Van der Bijl, 2014b). Na lista de substâncias proibidas pela AMA, o uso de designer steroids enquadrase na Classe S0, a qual abrange substâncias que não estão presentes em nenhuma outra categoria e que não têm aprovação de nenhuma autoridade regulamentar de saúde para uso terapêutico (Agência Mundial Antidopagem, 2019). ...
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Background: Nutritional supplements have received attention both from food manufacturers, as a means of marketing the added value to health; and from consumers, in terms of awareness, education, and improved health. To assist this process, it is important to have specific knowledge and understanding of the claims made on labels of nutritional supplement products used for general, and more specifically, for sports consumers. The industry is not regulated, and therefore the claims that are made may not always be accurate. Method: The aim was to describe the labelling and claims information on the labels of a select group of nutritional supplements, either manufactured in, or imported into South Africa. Specific predetermined categories of labelling and claims made on the containers were assessed and summarised. Results: Forty products were selected for analysis, of which 21 (53%) were locally assembled or manufactured products, and 19 (48%), international imported products. Ninety-five per cent of products contained a warning statement on the label. Eighty-five per cent of the nutritional supplement products had a disclaimer on the label. Ninety-eight per cent of the nutritional supplement product labels included some claim on the label. Conclusion: The following information, in particular, needs to be regulated and enforced as part of the labelling process, to ensure that the consumer can make an informed choice. This includes highlighting the potential for adverse events, encouraging warning statements pertaining to “exclusion of use, and “not a cure for disease states”, and alerting consumers of the potential for the presence of banned substances, based on laboratory screen methods.
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BACKGROUND. There is no clear distinction between the regulation of food, supplements and medicines in South Africa. Consequently, grey areas exist in implementing the legislation, particularly in the supplement industry. The increase in supplement sales in South Africa can be attributed to aggressive marketing by manufacturers whose claims are not always supported by published peer-reviewed evidence. Such claims often go unchecked, resulting in consumers being mislead about the role of supplements. As a result of poor regulation, contaminants or adulterants in supplements may also cause insidious effects unrelated to the listed ingredients. AIM. To assess the regulations, legislation, and claims associated with nutritional supplement products in South Africa. METHOD. Peer-reviewed literature and the relevant South African statutes were consulted. RESULTS. The National Health Act incorporates the Medicine Control Council, which is charged with ensuring the safety, quality and effectiveness of medicines, and related matters, including complementary/alternative medicines. The South African Institute for Drug-Free Sport and Amendment Act provides for testing athletes for using banned substances, but currently does not concern itself with monitoring nutritional supplements for contaminants or adulterants that may cause a positive drug test, which has implications for sports participants and also the health of the general population. The implementation of the Consumer Protection Act 68 of 2008 (CPA) could protect consumer rights if it is administered and resourced appropriately. CONCLUSION. The CPA should promote greater levels of policy development, regulatory enforcement, and consumer education of South Africa's supplement industry.
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