Scoping for doping

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Following the Athens Olympics, Henry Nicholls finds out if chemists are beginning to close on the athletes still determined to use performance-enhancing drugs?

There were more doping tests carried out at Athens than in any Olympic competition to date, the list of banned substances subject to testing has never been longer. By the time that the Olympic flame was extinguished on 29 August, 24 competitors had been found guilty of drugs offences. These achievements are down to the hard work of scientists behind the Olympic scenes determined to keep on the heels of the performance junkies. The gap between the drug takers and the drug testers is closing.

Drug taking in the Olympics is as old as the Games themselves. Olympic athletes in Ancient Greece used herbs and mushrooms to improve their athletic performance. And as the Athens Olympics came to a close more than 2000 years later, it is clear that athletes are as keen, probably keener than ever, to use performance-enhancing drugs to achieve fame and fortune. But now an unlikely competitor has entered the modern Olympic arena: the chemist. The athletes are usually first off the blocks, but with scientific cunning, the drug testers are never far off the pace.

The hub of the anti-doping operation at this year’s Olympic Games was at Greece’s new Doping Control Laboratory in Athens. The old laboratory was too small to cope with the hectic pace of analysis needed during the 16-day Olympic window. The new lab is 20 times larger and processed some 180 samples a day during the competition. ’We had to become 10 times faster in order to report the results in 24 hours,’ says lab director Costas Georgakopoulos. Depending on the sport, a urine sample might be subjected to up to 12 different analyses. To churn out this amount of chemistry, the number of scientists and technicians exploded from just seven before the Games to well over 100 during competition. Several of these came from anti-doping laboratories around the world. This kind of collaboration and exchange of ideas is crucial, says Georgakopoulos. ’This is the most important parameter in order to try to keep the distance between the cheaters and doping labs as close as possible,’ he says.

A spate of alarming drug-related deaths in the 1960s, mostly in cycling, highlighted the need for routine surveillance for drug abuse in sport. The first summer Olympics to benefit from testing were the 1968 Games in Mexico City; however, the only competitor to be caught out by the chemists that year was the Swedish pentathlete Hans-Gunnar Liljenvall, disqualified for being over the alcohol limit. By 1976, gas chromatography offered a reliable test for small molecules like anabolic steroids, and the number of doping disqualifications began to increase. In recent years, chemists have got to grips with testing for naturally produced hormones like testosterone and erythropoetin (EPO), and it looks likely that a reliable test for recombinant human growth hormone (hGH) will soon be up and running. With each anti-doping coup, the drug testers come one step closer to the drug takers. But as the gap closes, the would-be dope heads continue to search for new chemicals to give them a performance high. So the scientists must remain vigilant.

In 1999, the International Olympics Committee (IOC) set up an independent body - the World Anti-Doping Agency (WADA) - to coordinate anti-doping initiatives around the world. Last year, WADA activated its world anti-doping code, a detailed set of guidelines intended to harmonise procedure in the 32 dope-testing laboratories around the world that are recognised by WADA.

The largest of the anti-doping labs is the Olympic Analytical Laboratory at the University of California in Los Angeles, US. Set up in 1982, research in this laboratory has led to some of the most significant victories for the drug-testers over the takers. One of its early successes led to a drop in the abuse of testosterone and related hormones by sportsmen and women. It is relatively simple to detect such natural steroids, but because they are present in the urine of both doped and undoped male and female athletes, it is much harder to know whether the levels detected have been artificially elevated. The standard test had been to measure the ratio of testosterone relative to its cousin epitestosterone in a sample of urine. Normally, these steroids would be excreted in equal amounts, so a high level of testosterone relative to epitestosterone rings alarm bells.

However, the drug-takers nudged ahead of the testers again simply by taking epitestosterone to balance up the ratio. Then, in the early 1990s, the Olympic Analytical Lab came up with a test that distinguishes between natural and artificial testosterone. The chemical starting-point for synthetic testosterone is diosgenin, which is obtained from yams. This plant-based origin means that synthetic testosterone has a different carbon isotopic signature from testosterone produced and secreted by animals. As soon as this test was derived, the drug takers had to move on to other ways of enhancing performance that were still beyond detection.

For a while, synthetically produced recombinant EPO became the drug of the moment. The body makes EPO naturally in the kidneys, and its effect is to boost the number of red blood cells, thereby increasing the amount of oxygen available to muscles. When recombinant EPO appeared on the medical market in the late 1980s, used mainly for treating anaemia or for patients on dialysis, the wannabe drug-takers were quick off the mark. In 1990, the IOC added EPO to its list of banned substances. But a test that could distinguish between natural and recombinant EPO did not appear for 10 years, and in that time, the hormone was implicated in the deaths of several world-class cyclists; the suspicion was that they were taking so much EPO that their blood became too thick to flow.

In 1999, scientists at the Australian Institute of Sport (AIS) and the Australian Sports Drug Testing Laboratory worked out that synthetic EPO affects the blood in predictably different ways than does the natural form of the hormone. Then in 2000, just in time for the Sydney Olympics, researchers at the WADA-approved lab in France devised a test that could identify synthetic EPO in urine.

Naturally produced EPO is an acidic glycoprotein with four N-linked and one O-linked sugars. Although recombinant versions of the hormone all have the same amino acid sequence, the sugars they carry have different charges, which means they are more alkaline than natural EPO. Therefore, natural and synthetic EPO can be distinguished on the basis of their isoelectric points - the pH at which a protein has an equal number of positive and negative charges. Using this technique, an assay of frozen urine samples from the infamous 1998 Tour de France found that more than one in eight competitors had been using recombinant EPO. At Sydney, several athletes tested positive before the Games and withdrew. It was time for the drug takers to go in search of a new fix.

Last year, the Olympic Analytical Laboratory at UCLA closed in on the drug takers once more. This time, they discovered an entirely novel steroid: tetrahydrogestrinone (THG), which had all the hallmarks of being designed specifically to avoid detection. Indeed, it might never have been uncovered, but for one fortuitous event. In June 2003, a spent syringe, which allegedly contained an anabolic androgenic steroid, was posted anonymously to the Los Angeles laboratory.

Initial analysis of the sample using GC/MS produced a string of confusing peaks, most of which did not correspond to known anabolic steroids. There was, however, one peak they identified as norbolethone, which appeared to be present in small amounts, suggesting that the mystery chemical might have the same carbon skeleton. From high-res GC/MS, they deduced that the formula of the compound had to be C₂₁H₂₈O₂. Mass spectra also revealed fragments similar to those produced by the steroid gestrinone, a drug for endometriosis that is on WADA’s list of banned substances. These were just enough clues to allow the scientists to have a stab at the structure of the unknown steroid.

Scientists synthesised THG from a spent syringe

They then attempted to make the steroid afresh, synthesising it by tagging four hydrogen atoms onto the D-ring of gestrinone. GC and LC/MS/MS of the product produced identical traces to the steroid from the syringe, confirming that their hunch had been right. The test that emerged was then used on urine samples from athletes, revealing that abuse of THG was widespread.

So with the drug testers drawing level with the drug takers over an array of anabolic steroids, synthetic hormones like testosterone and EPO, and the odd ’designer steroid’ such as THG, is it possible to win the race? The switch from chemistry to biology is the big challenge, says Steve Maynard, head of drug surveillance at HFL, a Cambridgeshire, UK-based drug testing company that is the latest lab to receive WADA approval. The chemists must anticipate what the new methods of doping will be, he says.

One of the biological substances that is high up on WADA’s hit-list is human growth hormone (hGH). In 1992, an anonymous survey found that one in 20 American high-school students had used recombinant hGH to build muscle and athletes have been caught with ampoules of the hormone in their possession. Although hGH is on WADA’s list of banned substances, there is still no approved test that can demonstrate its use. The problem is that once it enters the bloodstream, hGH is broken down within 20 minutes. Its secretion from the pituitary is also affected by sleep, nutritional status, exercise and stress, so can fluctuate wildly during the day. Nevertheless, there are two approaches that could show up abuse of hGH.

One way is to look for the molecular consequences of artificially elevated hGH, says Cathy McHugh, an endocrinologist at Southampton University, UK. hGH causes an increase in the levels of several proteins, and two of them - insulin-like growth factor-I (IGF-I) and procollagen 3 - are of particular interest because they remain elevated long after hGH has been broken down. This opens the window for detection, says McHugh, who has recruited more than 200 athletes to study the natural variation of these proteins in the circulation immediately after exercise. One drawback is that the normal levels of IGF-I and procollagen 3 are different for each individual, so it may be necessary to take several measurements from the same athlete to create a profile of how their hormones fluctuate, she says. ’You have to know what’s normal before you say that something’s abnormal.’

Alternatively, a more direct approach is being developed in Germany by Christian Strasburger, professor of clinical endocrinology at Charit? Universit?tsmedizin in Berlin. Unfortunately, the ¹³C/¹²C isotopic ratios of endogenous and recombinant hGH are too similar to be useful. Nor are there glycosylation sites as there are for EPO that can be used to form the basis of a test. However, the pituitary gland does secrete many different forms of hGH - most are 22 kDa in weight, but 20-kDa hGH is also released along with smaller peptide fragments. By contrast, recombinant hGH comes in only one form - the 22 kDa variety. A sudden injection of recombinant hGH therefore increases the amount of the 22-kDa isoform relative to the other naturally produced varieties, and crucially hGH in the bloodstream exerts negative feedback on further secretion of the hormone from the pituitary gland, further exaggerating the bias towards 22-kDa hGH. It is possible to detect this change to the isoform makeup in the blood using monoclonal antibodies with different affinities to the different hGH isoforms.

This year, Strasburger has demonstrated the procedure to scientists at the WADA-accredited laboratories in Dresden, Cologne, London, Sydney and Athens. These labs have all the necessary equipment, reagents and expertise to conduct this test, he says. This raises the possibility that blood samples from this year’s Olympics have been tested for recombinant hGH. ’It may already have happened,’ says Strasburger.

But the big biological concern is that athletes might soon begin gene doping - using gene therapy to boost performance. This avenue has been opened up by recent advances in medical research. For example, patients with Duchenne muscular dystrophy inherit a genetic mutation that leaves them without dystrophin - a shock-absorbing protein that protects muscle fibres when they move. The result is that muscle fibres die and are largely replaced by fibrous tissue and fat. Gene therapy has the potential to smuggle a functioning copy of the dystrophin gene into muscle cells of such patients, and human trials are being planned. However, unscrupulous athletes could easily use this technology to improve the performance of their muscles.

Similarly, there is research into gene therapy to reduce wasting of elderly muscles. With aging, muscle-repair mechanisms start to pack up. But a dose of IGF-I to the muscles stimulates the satellite cells surrounding muscle fibres to manufacture the proteins that are needed to rebuild muscle. Experiments in rats show that IGF-I gene therapy to muscles followed by exercise increases strength by nearly twice that of just training alone. Human trials may be as much as a decade away, but an athlete could easily experiment with this kind of emerging treatment.

To keep up with these threats to drug-free sport, the IOC and WADA are now putting money into laboratories to develop a test for gene doping. HFL is one such laboratory. ’We are constantly investing in research to develop new methods,’ says Maynard. However, it is unlikely that the drug testers will ever be able to break into the lead and surge ahead of the drug takers, he says. The Olympian race between the drug takers and drug testers looks set to run and run.