Catching the dope cheats

How do labs keep up with the ever more sophisticated techniques that athletes use to try and cheat the system?

The Tour de France of summer 1998, was dubbed the Tour of Shame after the arrest of Willy Voet, one of the signeurs for the Festina cycling team, for the possession of illegal prescription drugs including narcotics, erythropoietin (EPO), growth hormones, testosterone, and amphatamines. Two weeks later, following a raid, police found further doping products in the possession of the TVM team. Further raids were threatened, and after talks and a mass exodus of cyclists, the Tour resumed. However, the image of competitive sporting was to change forever.

Eight years on and the Tour is still haunted by doping allegations. In May 2006 Lance Armstrong, the American cyclist with 6 consecutive Tour victories to his name, was formally cleared by independent Dutch investigators of doping during the 1999 Tour de France. A French newspaper had claimed that Armstrong’s urine samples during the 1999 tour had later tested positive for the endurance-boosting hormone EPO after they were retested in 2004. Following an investigation by the International Cycling Union into the handling of the urine tests by the French national anti-doping laboratory, known as LNDD, it was found that the LNDD had behaved in ways that are completely inconsistent with the rules and regulations of international anti-doping control testing, thereby exonerating Armstrong. According to the Dutch judge appointed during the investigation, the way in which the positive results were obtained is very different from the analysis procedure required by the World Anti Doping Agency, and that the results do not even qualify as a finding. It appears that the urine samples had been used in research programmes.

However, it is not only the high profile sports competitions which are dogged by allegations of doping. The International Olympic Committee has recently banned the consumption of beta inhibitors in precision sports such as chess, motor racing, billiards, bowling, air sports, freestyle winter sports, gymnastics, wrestling, motorcycling, modern pentathlon and sailing.

Beta inhibitors are medicines that regulate the heart pace, slow down cardiac frequency and stimulate the attention. They cause a reduction in improvement of pulse (reducing trembling), and they also have an anti-stress effect. Beta inhibitors include propanolol, acebutonol, alprenolol, labetalol, metoprolol, nadolol, oxprenolol and sotalol.

Until now different techniques, including flourimetry and phosphorimetry, have been used to determine propanolol in biological fluids, for example in urine samples. These methods function on a batch system and require various tedious preliminary procedures such as pre-concentration in an organic solvent because they can be tested.

Researchers at the University of Granada, Spain, have recently developed a new technique for the rapid detection of propanolol in urine samples. The method makes use of analytical sensors that allows the determination the presence of propanolol in just a few minutes and with an accuracy of 0.2?g/l.

The analytical advantages of optical sensors are well documented. Combining flow-injection analysis techniques with detection on optically active surfaces with an immobilised indicator packed in a flow-through cell, called ‘optosensing flow-injection analysis’, has proved to offer important advantages because of its high sensitivity, selectivity, precision, simplicity, speed and low cost. This technique has led to shorter turnaround analysis time and reduces cost for doping controls. As a large part of the samples prove to be non-doped, rapid analytical methods that provide reliable ‘yes’ or ‘no’ results are of increasing interest. These tests can usually be described as systems that ‘filter’ samples to select those with analyte content levels similar to or higher than a previously established threshold. These ‘probably doped’ samples will then need to be examined with more exact instrumentations, but doubtless screening will have been far more rapid.

The detection of beta inhibitors is not the only area which has sparked interest in the anti-doping world. Testosterone is particularly difficult to detect because there is no chemical difference between the exogenous and endogenous testosterone, and you cannot reply on testosterone levels because there is a lot of natural variation. The current standard test replies on the testosterone/epitestosterone ratio, but there is a lot of variation there too and athletes can evade it by taking both testosterone and epitestosterone.

A technique proposed recently by researchers relies on the isotope ration of carbon 12 (C-12) and carbon 13 (C-13). The isotope ratio is determined by diet and again is different for each individual. However, by comparing the C-12/C-13 ratio in testosterone to that of other precursors (for example cholesterol), it can be determined whether the extra testosterone is exogenous.

To make this technique work, separation of the various compounds using gas chromatography is first needed. Mass spectroscopy is then used to determine the isotope ratios of the various fractions. Unfortunately the technique needed further fine-tuning as the reactive groups on the steroids tend to react with the GC column, thereby giving inaccurate results. It has been suggested that hydropyrolysis can protect these reactive groups without changing the carbon ratios, thereby potentially rendering this technique valid.

Despite the efforts by the anti-doping brigade to develop more sophisticated and accurate methods of analysis, the overall research community is one step ahead, and these days the doping market has moved into the genetic arena. Gene doping has been defined by the World Anti Doping Agency as ‘the non-therapeutic use of cells, genes, genetic elements, or of the modulation of gene expression, having the capacity to improve athletic performance’. As far back as 2003, the Prohibited List of Substances and Methods (WADA) was amended to include gene doping as a prohibited method.

An example of gene doping would involve gene therapies to treat muscle-wasting disorders. Among these are therapies that give patients a synthetic gene, which can last for years, producing high amounts of naturally occurring muscle-building hormones such as insulin-like growth factor I (IGF-1). The chemicals are indistinguishable from their natural counterparts and are only generated locally in the affected tissue. Nothing unusual would enter the bloodstream, so officials will have nothing to detect in a blood or urine test.

The World Anti-Doping Agency (WADA) has already asked scientists to help find ways to prevent gene therapy from becoming the newest means of doping. In December 2005 the WADA hosted its second landmark meeting on gene doping, which took place in Stockholm. At this meeting, delegates drafted a declaration on gene doping which, for the first time, included a strong discouragement on the use of genetic testing for performance. The first product to be associated with genetic doping emerged during the Torino 2006 Olympic Winter Games, where Repoxygen was discussed as a possible substance in use at the Games. Repoxygen, developed by UK firm Oxford Biomedics, delivers the gene for erythropoietin to muscle cells in a vector configuration, bringing the gene under the control of an oxygen-sensitive gene switch.

Since January 2005, WADA has funded a series of projects which aim to develop systems for the detection of gene doping. The most recent project is being carried out by HFL Laboratory Inc in Fordham, Cambridgeshire. The project looks at the application of cellular chemistry and proteomic approaches for the detection of gene doping, which proposes a more global approach for the detection of doping. Following doping with doping substances or the use of genetic manipulation, the expression of one or more genes and/or proteins will be altered in several accessible tissues such as blood cells or buccal mucosa cells. These changes in gene/protein expression will be detected through the application of high performance transcriptomic or proteomic techniques. Ultimately this will lead to the identification of abnormal RNA/protein patterns, representing molecular signatures associated to the use of doping substances, such as IGF-1 or growth hormone. Another project, being carried out at the Pharmacology Research Unit at the Institut Municipal d’investigacio Medica, Barcelona, is researching the non-invasive molecular imaging of gene expression useful for doping control. This is currently a pilot study in animals after erythropoietin gene transfer. In this project, imaging will be used to detect the RNA being formed in unusual tissues after the gene transfer process. This approach is applicable to any gene transfected to tissues not usually expressing the ‘doping’ protein, such as muscle for EPO. Imaging of mRNA will be carried out by the use of antisense peptide nucleic acids oligonucleotide probes labelled for tomographic detection. The pilot project is being carried out to image the presence of transfected EPO genes into the muscle of mice.

By Maria Anguita. Maria is a freelance science and health writer and holds a degree in biochemistry with medical biochemistry.