EARLY in February runners turned out for a cold, four-mile race round the
city of Derby. It was a low-key event, with a high-profile winner鈥擝ritish
Olympic hopeful Diane Modahl. The event was Modahl鈥檚 first since August 1994,
when she was charged with taking the hormone testosterone and banned from
competition by the British Athletic Federation (BAF). And although the BAF
cleared her on appeal in July, Modahl鈥檚 case still has some distance to run.
Next week, the BAF鈥檚 parent organisation, the International Amateur Athletic
Federation (IAAF), will meet to decide whether to re-try the case. If it does,
Modahl could find herself banned once again.
At last summer鈥檚 appeal, Modahl鈥檚 defence team said that the extraordinarily
high level of testosterone in her urine could have come from bacteria in the
samples. To back up their claim they presented the results of a controversial
experiment which showed that testosterone levels could rise in urine collected
from women who had just exercised if the samples were not properly stored. The
IAAF鈥檚 decision next week will hang on its own experts鈥� view of that experiment,
and on new evidence dealing with the biochemical behaviour of bacteria in
urine.
One of the questions facing the federation is why scientists at laboratories
accredited by the IAAF鈥檚 sister organisation, the International Olympic
Committee (IOC), cannot replicate the original urine experiment, while
researchers on the defence team can. The dispute is calling into question the
procedures that are meant to catch athletes who take testosterone. According to
the defence team, no one can be sure that a competitor who tests positive has
taken the banned drug or whether someone who tests negative has not. In an
Olympic year, officials are under pressure to resolve the argument over the
tests.
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Modahl鈥檚 problems started in Lisbon in June 1994. After running
in an 800-metre race, she was asked to provide a routine
urine sample. Attendants at the testing centre divided her sample between two
bottles, 鈥淎鈥� and 鈥淏鈥�, ready to be taken to the local IOC-accredited laboratory.
Two months later, while waiting to compete in the Commonwealth Games in Canada,
Modahl was notified that her urine sample contained an abnormally high level of
testosterone. She was sent home in disgrace.
A week later tests by the Lisbon laboratory on the 鈥淏鈥� sample appeared to
confirm the original result. According to those tests, Modahl鈥檚 testosterone
level was 120 nanograms per millilitre, one of the highest ever recorded in a
female athlete. The average for a women is around 10 ng/ml. In accordance with
IOC and IAAF rules, the Lisbon laboratory also compared the urine level of
testosterone with that of another steroid called epitestosterone, a comparison
that compensates for more or less dilute urine. Modahl鈥檚 testosterone to
epitestosterone (T/E) ratio was more than 40, way higher than the IOC鈥檚
permitted limit of 6 for both male and female athletes. These figures led the
BAF disciplinary hearing to punish Modahl with a humiliating four-year
ban.
But even before that hearing, evidence was emerging that made the case
against Modahl look less than watertight. The urine samples taken in Lisbon had
spent two nights and a hot summer鈥檚 day unrefrigerated before they were analysed.
When the seal on the 鈥淏鈥� sample was broken, the smell of ammonia was so
strong that David Cowan, the director of the IOC-accredited laboratory in London
who had flown to Lisbon to observe the tests, asked that the pH be
measured. The sample was alkaline, with a pH of 8.85, which is
generally accepted to be a sign of bacterial degradation.
Reasonable doubt
These factors helped float the theory that would eventually tilt the appeal
in Modahl鈥檚 favour. Modahl had consulted the endocrinologist John Honour of
University College London Hospitals to find out whether she had a medical
condition that would produce high levels of testosterone. Honour said she did
not. But when he scrutinised the Portuguese analysis of the steroids in Modahl鈥檚
urine, he was struck by what he saw as a glaring inconsistency. 鈥淚t was
strange,鈥� says Honour. 鈥淭he very scientific evidence that was being used to
prove her guilt, to me proved her innocence.鈥�
Honour noticed that the levels of the steroids androsterone and aetiocholanolone
were extremely low even for a normal person, let alone someone who was
supposedly taking testosterone. 鈥淲hen a person takes testosterone it is partly
metabolised into these two,鈥� he says. 鈥淭he levels should have been very high.鈥�
To Honour, the pattern suggested that some of the numerous bacteria that live on
or in the human body had transformed the two hormones back into
testosterone.
Modahl鈥檚 solicitor, Tony Morton-Hooper of the London firm Mishcon De Reya,
recruited Honour to the defence team, followed by three biochemists: Rod Bilton
of John Moores University in Liverpool, Paul Talalay of Johns Hopkins University
in Baltimore and Robert Owen of the German Cancer Research Centre in Heidelberg.
They put together a detailed hypothesis of how bacteria in the urine break down
steroids and produce testosterone.
But it was the fifth scientist on the team, Simon Gaskell, professor of mass
spectrometry at the University of Manchester Institute of Science and
Technology, who supplied the clincher. He took urine samples from two women
after they had exercised, split each sample, and stored one half at 鈭�20 掳C and
the other at 37 掳C for three days. The T/E ratio of both the samples that were
kept at the higher temperature increased, one to more than 12. 鈥淭he experiments
established unequivocally that the T/E ratio could be raised by inappropriate
storage,鈥� he says.
Faced with this experimental evidence, the appeal panel ruled in Modahl鈥檚
favour. 鈥淲e cannot be sure beyond reasonable doubt of Mrs Modahl鈥檚 guilt,鈥� said
Robert Reid, the barrister who chaired the appeal. Modahl was free to race
again鈥攁t least temporarily.
But Reid also cautioned against over-interpreting Gaskell鈥檚 result鈥攁 warning
that the IAAF appeared to take to heart. Five days after the appeal, it
announced that it would refer the case to an arbitration panel and ask the
Lisbon scientists to re-confirm their result.
The IAAF鈥檚 decision has spurred the defence scientists to add flesh to their
theory that bacterial action led to the high testosterone levels in the Lisbon
urine sample. According to Bilton, what happens is this. First, bacteria such as
Proteus mirabilis鈥攁 species that lives happily in the human gut and so
is not unlikely to contaminate a urine sample鈥攑roduce the enzyme urease, which
converts the urea in urine into ammonia. That makes the urine sample alkaline
and rather hospitable to other bacteria species that can transform urinary
steroids鈥攊ncluding, in theory at least, androsterone and aetiocholanolone鈥攊nto
testosterone.
Athletic bacteria
Bilton doesn鈥檛 know exactly which species are involved. 鈥淭here are too many
to go into,鈥� he says. 鈥淏ut Escherichia coli and Klebsiella
pneumoniae are probably the most likely.鈥� Both species thrive at a
pH between 8 and 9, and microbiologist Reuben Gr眉neberg at
University College London Hospitals has cultured the two species from Modahl鈥檚
urine. Bilton says he has new data which show that a dehydrogenase enzyme
produced by both Klebsiella and E. coli transforms a steroid
androst-5-ene-3beta,17beta-diol, that is found in urine, into a testosterone
precursor. At a high pH鈥攕uch as that of Modahl鈥檚 urine鈥攖his chemical
immediately switches to testosterone.
Owen, too, has data ready for the IAAF. He has shown that three Klebsiella
enzymes convert a steroid called dehydroepiandrosterone sulphate, also present in urine,
into testosterone in four easy steps.
More importantly, Bilton has reproduced Gaskell鈥檚 experiment. After
incubating urine from three female athletes for 48 hours at 30 掳C, he recorded
testosterone levels of between 120 and 150 ng/ml. He is waiting for the
all-important T/E ratio to be measured.
But others have not been so successful, including several scientists from
IOC-accredited laboratories. Christiane Ayotte of the IOC-accredited laboratory
in Montreal has looked at numerous poorly stored urine samples collected from
competitors over the last five years. 鈥淚 can swear to you that we have never
observed the formation of testosterone,鈥� she says. 鈥淥n the contrary, we have
just observed its degradation.鈥� In a forthcoming issue of the Journal of
Chromatography, Ayotte will report just such cases, in which bacteria have
caused the levels of testosterone, and the T/E ratio, to fall.
This work raises the spectre of athletes who have taken testosterone getting
the all-clear on their dope tests, though Ayotte argues that there is a way
round the problem. Steroids produced by the human body and injected or ingested
steroids are usually bound to a sugar molecule. By contrast, steroids produced
by bacteria are initially in their free, unbound form, and then gradually become
oxidised. Ayotte conducts an additional test that detects the free form and the
oxidation products. If their levels are too high, she discards the
sample.
Bilton agrees with Ayotte that urine testosterone levels can, and do, fall.
鈥淚鈥檓 not saying that every time you get bacterial growth you get testosterone,鈥�
he says. 鈥淚t鈥檚 entirely capricious.鈥� It depends, he says, on many variables such
as the type of bacteria that are present, the temperature the sample is stored
at, the availability of oxygen, and so on. Indeed, it is the variability of
these conditions that he uses to rebut a question that has been levelled against
the defence鈥檚 case: why haven鈥檛 high testosterone levels been detected more
often in the urine of female athletes? Bilton says that from his experiments it
appears far more likely that bacteria will reduce testosterone levels and
generate false negative test results, than raise testosterone levels and
generate false positives. 鈥淵ou need very specific conditions to get a rise,鈥� he
says.
鈥淭he whole business of analysing spoilt urine is unsound. It鈥檚 a terrible
morass,鈥� says Bilton. Unlike Ayotte, he believes that once a urine sample has
spoilt, it can never safely be used for analysis. Bilton and scientists close to
the IAAF contend that it is essential to stop samples degrading in the first
place.
In fact, the IOC鈥檚 guidelines recommend that samples with a pH
higher than 8.2 should not be tested. Cowan told the British appeal panel that
he would not have analysed Modahl鈥檚 urine sample because its pH was
above this limit. Morton-Hooper made much of this point at the appeal. He also
stressed other deviations from the guidelines. The Lisbon laboratory never
measured the pH of Modahl鈥檚 鈥淎鈥� sample, for example, and there were
flaws in the 鈥渃hain of custody鈥� for her samples. Arne Ljungqvist, chairman of
the IAAF鈥檚 doping commission, argues that these small anomalies do not matter.
杏吧原创s at the Lisbon laboratory refused to answer New 杏吧原创鈥檚
questions 鈥渇or legal reasons鈥�.
But one thing is clear: the IAAF has a difficult decision to make at next
week鈥檚 meeting. The Lisbon scientists have refused to conduct a third test on
Modahl鈥檚 urine, and so now the IAAF must decide whether or not to go ahead with
arbitration anyway. If it does, the IAAF will have to prove beyond reasonable
doubt that Modahl took testosterone. But if it decides to drop its opposition
to the British appeal panel鈥檚 decision, it will open the door to other athletes
trying the same defence.
Meanwhile, in an unusual move, the IAAF told Modahl that she could run again
in international competitions. But if the IAAF goes for arbitration, and it
finds that the BAF appeal panel was wrong, the international ban will come into
force again.
Modahl isn鈥檛 speaking to the press, but her husband Vincente Modahl, who is
also her trainer, says the IAAF has wasted too much of one runner鈥檚 career. The
scientists should sort out their problems on their own time, he says. 鈥淚t
shouldn鈥檛 be Diane that pays.鈥�