The Dope On Testosterone Test

NEWS ANALYSIS

Now that the initial media feeding frenzy over the Floyd Landis Tour de France doping scandal has abated, it's time for a sober perspective on doping in sports. This opportunity presents itself every couple of years when a new round of these scandals fuels newspaper headlines. The stories under the headlines often are based on hearsay and speculation, and they almost always are light on science. This combination can lead to a lot of head-scratching while one tries to make sense of what is going on and in this case what, if anything, will happen to Landis.

To recap, Landis won the world's premier cycling event last month, in part because of an impressive performance in one of the mountainous stages near the end of the Tour in which he pulled away for a solo victory. The urine sample he was required to give following that stage win on July 20 subsequently tested positive for an abnormal amount of testosterone. Landis was subject to eight drug tests during the Tour this year. A few of the tests were administered before the positive test, and a few came afterward. Only the one sample came back positive for a banned substance.

Urine and blood tests are analyzed for different sets of performance-enhancing drugs or agents to cover up drug use. For simplicity, the discussion that follows will focus on urinalysis for testosterone. When an athlete provides a urine sample, he or she divides it into A and B portions. The two bottles are sealed in the presence of the athlete, and a strict chain of custody is followed to transport the samples to a lab. For the Tour de France, the French national doping-control lab in Châtenay-Malabry, near Paris, carried out all the tests.

The A portion is tested, and if a positive finding is made, it is retested for confirmation. The retest is usually done by a different technician and on a different and generally more sophisticated instrument to rule out human error. If the A test is confirmed positive, the B portion of the sample is also tested. The result of the B sample is considered the final result, and it almost always confirms the result of the A sample. If the B sample is negative, however, the positive A result is thrown out.

Athletes who test positive then face an adjudication hearing to determine their guilt or innocence. Just because an athlete has a positive test doesn't mean he or she is guilty of a doping offense. Extenuating circumstances are taken into consideration, and quite often test results are overruled, either at the hearing or later on appeal. Landis is currently waiting for his hearing, which will be administered by the U.S. Anti-Doping Agency because he is a U.S. citizen. The process could take several months to complete.

The testing process up to this point is supposed to remain confidential, according to procedures in the World Anti-Doping Code. In the Landis case, cycling authorities first announced on July 26 that one Tour rider had an "adverse analytical finding," a permissible announcement by the rules. What was not okay was a premature announcement by Landis' team the next day that he had a positive test, along with leaks to reporters about the details of the testing from unnamed sources "with knowledge of the results" at the international cycling governing body. Coupled together, these breaches of protocol fueled speculation about how Landis could have tested positive for testosterone and has kept him backpedaling, trying to explain himself.

The cutoff for a positive test for testosterone, set out in the antidoping code, is a testosterone to epitestosterone ratio (T/E) of 4:1 or higher. Testosterone is an anabolic steroid well-known as the male sex hormone, and it has a number of medical uses. Epitestosterone, as its name suggests, is an epimer of testosterone-a stereoisomer that differs only in the configuration at one chiral center, which in this case is the hydroxyl group at C-17 of the steroid ring system.

That small structural difference leads to a large physiological difference. Epitestosterone doesn't have the anabolic—that is, muscle-enhancing—properties of testosterone, and its role in humans is unclear. One possibility is that epitestosterone serves as a foil to moderate testosterone binding to androgen receptors, particularly in the years before puberty.

Levels of testosterone and epitestosterone are age-dependent, and they sometimes can vary widely from one individual to another. But because the compounds are produced by different biosynthetic pathways, their relative levels are independent and a ratio of the two compounds measured by mass spectrometry can be used as a good doping-control test, rather than relying on a direct measurement of testosterone.

The natural T/E ratio for adult men on average is about 1:1, and it rarely exceeds 4:1. Some physicians quoted in media reports about the Landis case say that higher natural ratios, as high as 8:1 or 10:1, are possible. Landis reportedly had a ratio of 11:1, which at face value looks pretty incriminating.

Anabolic steroids typically are taken in several cycles of weeks to months to increase muscle mass and strength. The one-time spike in Landis' testosterone level is puzzling, in part because cycling isn't a power sport like sprinting or hitting a baseball. In fact, most world-class cyclists, including Landis, tend to be average-to-tall skinny guys you wouldn't expect to see on the cover of a muscle magazine. As a side note, leading U.S. sprinters Justin Gatlin (testosterone) and Marion Jones (erythropoietin) recently had positive drug tests, and Major League Baseball player Barry Bonds is still being investigated for his possible role in a steroid distribution conspiracy.

Another performance-enhancing benefit of steroid use is that it allows an athlete to recover more quickly from hard training sessions and thus train harder for longer periods of time. Some nonpower-sport athletes, like distance runners and swimmers, have been reported in the clinical literature to take small doses of testosterone as a prophylactic measure to counter the effects of training and competition without building much muscle. It's pretty certain, though, that a sudden artificial boost would not provide an immediate performance boost.

So why would Landis start taking testosterone in the middle of the Tour, as his drug test indicates, when he would not gain much benefit from it and he knew he would be tested repeatedly? Landis answered that question by strongly denying he took any testosterone. His denial opened the door to a litany of other possible physiological effects that could explain a false-positive test.

First of all, Landis could naturally have an abnormally high T/E ratio. This fact will be easily determined during the hearing by checking the results of his other urine tests. Landis also initially stated that the abnormal result could have come from his drinking alcohol the night before the test, dehydration that concentrated testosterone in his urine, thyroid medication for hypothyroidism, cortisone injections he was allowed to have under a medical waiver to relieve inflammation associated with a degenerative hip joint, or a combination of these factors. Some clinical research suggests the extraordinary effort in his mountain stage win could have given him a temporary natural testosterone boost.

Despite the speculation about its origin, the one thing certain about the high T/E ratio is that it prompted the doping-control lab to pursue the matter further and retest the sample by gas chromatography/combustion/isotope-ratio mass spectrometry, which is considered foolproof for identifying synthetic testosterone. This method determines the percent difference between the ratio of ¹³C to ¹²C in CO₂ from the combustion of testosterone or its metabolites in the urine sample and the ¹³C to ¹²C ratio of an internal CO₂ standard. When synthetic testosterone is used, the percent difference is reduced, by a few percent, relative to baseline isotope ratios of natural steroids, their precursors, and their metabolites in urine. According to doping-control authorities, Landis flunked the test.

The isotope-ratio test is possible because synthesized steroids are made from starting compounds (diosgenin or stigmasterol) obtained from soybean or other plant oils. Soybeans and most agricultural crops are C₃ plants, which means they first form a three-carbon molecule (3-phosphoglyceric acid) from CO₂ during photosynthesis. Other plants that are integral to the human diet, such as corn and sugarcane, are C₄ plants, which first form four-carbon molecules (oxaloacetate, malate, and aspartate). Because the human diet consists of a mix of C₃ and C₄ plants, naturally produced steroids will have a slightly higher amount of ¹³C than synthetically derived steroids, hence the decrease in the percent difference observed for a positive testosterone doping test.

Landis has added another dimension to the scandal by suggesting that circumstantial evidence points to there being something sinister at work-that is, someone tampered with his sample, rigged the analysis, or altered the results. Indeed, the French drug-testing lab and officials at the international cycling federation have been under the gun before for improperly handling samples and quietly providing documentation on test results to reporters.

Adding weight to that argument is that Landis was the only rider, out of a starting field of 176 cyclists, to have a reported positive test during the Tour. Cycling officials have explained that away by saying the cheaters were excluded from the Tour de France because they had been caught up in a related Spanish doping scandal earlier in the year. That episode led to nine riders, including several favorites, being banned from this year's race.

As one commentator in the press noted, Landis may now be paying for the past sins of other cyclists. Most people are probably hoping that he is innocent. Still, it's hard to forget that cycling has proven time after time to be a drug-ridden sport, as have most professional and international sports where athletes earn large sums of money to simply play. One possible deterrent, which is starting to be used, is to force athletes to return money if they are caught doping.

The Floyd Landis story is far from over. The U.S. Anti-Doping Agency will ultimately determine what happens next. It will decide whether Landis broke the rules, no matter what the reported positive test results might indicate. If Landis is found guilty, he would lose his Tour de France title (he is still the champion for now) and be banned from competing for two years or more. But the ultimate punishment, already invoked, is permanent damage to his career as a cyclist and his reputation, even if he is exonerated.

If, however, Landis is found not to have broken the rules, the public should not forget that the scientists conducting the doping tests are merely fact-finders. It's up to the sports-governing bodies, which include scientists on their review panels, to decide what to do with the facts. It's up to federal governments to more tightly regulate sports and enforce more severe punishments for violations.

For chemists and other scientists, there are many opportunities for further research to better understand human physiology, particularly the role of steroids, and for developing better tests to detect doping. It's a good alternative to sitting in an armchair watching doping scandals unfold on TV.