Doping in sport: consequences for health,clinicians and laboratories

The full extent of doping in sport and exercise is unknown. Drugs are often used to increase muscle mass (anabolic drugs), decrease fat muscle (lipolytic drugs) or increase oxygen delivery to tissues. They have also been used to improve mood, steadiness, fatigue, confidence, euphoria, tolerance to pain, relaxation, alertness, concentration and reaction time. Diuresis to bring about a reduction in body weight is well recognised. The review by Bird, Greaves and Burke ¹ covers the history of doping in sport and, importantly, focuses on the manifold health risks: acne, virilisation, deepening of the voice, infertility, subdural haematomas, tendon injuries, altered liver and kidney function, peripheral oedema, cardiac hypertrophy, myocardial ischaemia and thrombosis. Some of these are irreversible and many drugs pose health risks including cardiovascular disease and death. Some drugs are taken to counteract the side-effects of the abused drugs. For example, aromatase inhibitors and selective oestrogens receptor modulators (SERMs) are sometimes taken to mask the gynaecomastia caused by anabolic steroid abuse.

Doping control was introduced in 1968 by the International Olympic Committee (IOC). Initially amphetamines were the target of testing; anabolic steroids were added in 1974. Subsequently a wide range of drugs and drug groups have been added: beta-blockers, sympathomimetics, diuretics, narcotics, growth hormone, glucocorticosteroids and erythropoeitin. In recent years tests have been developed for dextrans, insulins, adiponectin receptor agonists, mechano-growth factor (MGF), selective androgen receptor modulators (SARMs), peroxisome proliferator activated receptor (PPAR) gamma agonists, sirtuin activating compounds, erythropoiesis stimulating agents, gonadotrophins and releasing hormones.

Initially, drug tests set out to detect pharmaceuticals which do not occur in the body. However, the detection of abuse of natural substances has been a challenge for the doping laboratories. “Natural” hormones can be produced commercially by chemical and in vitro techniques for recombinant proteins. Also, since drug testing has largely used urine samples, abuse of natural substances cannot be reliably detected from an increased concentration in the sample. Moreover, adulteration of samples with chemicals and enzymes is an additional problem. Tests therefore have been developed for metabolites and for disturbances of a hormonal axis. An additional approach has been to look for altered isotope ratios. For example, steroids for human use are produced from plant sterols. These have different carbon 12/carbon 13 isotope ratios from endogenous human steroids, allowing this difference to be exploited by testing.

Drug testing in sport was originally based exclusively on collection of samples immediately after a race or competition, but awareness of this possibility meant that illicit use of performance-enhancing substances was timed so that the predictable timing of sampling coincided with a drug-free period (in contemporary parlance, the athlete had stopped ‘glowing’ by the time of sample collection). Unannounced (i.e. minimal warning) out-of-competition testing is therefore used in addition. Another comparatively recent development has been the introduction of long-term blood monitoring of various haematological parameters (providing the so-called athlete’s ‘biological passport’). Changes in the parameters may indicate drug abuse, warranting additional confirmatory testing. This approach was devised initially to detect abuse of erythropoetin and growth hormone; it now includes steroids.

The effects of drugs used in sport on health may present a challenge for doctors and clinical biochemists; most doctors have little experience of how these present. The clinical biochemist needs to be alert for unusual results that may be the result of ‘sports drug abuse’: unusual results in electrolytes, uric acid, cholesterol, triglycerides, haemoglobin, haematocrit, clotting factors, liver function, sex steroids, pituitary hormones, sex hormone binding globulin, glucose, insulin and insulin-like growth factor. These changes are helpfully tabulated in the review by Bird et al. Assay interferences are also possible. Samples for drug testing in sport are not preserved during the time between collection and receipt at the laboratory. This probably stems from the forensic background of many laboratories involved in this kind of testing. Tests have been introduced that are said to recognise a signature of bacterial degradation.

In the medical setting an athlete should be questioned about use of nutritional supplements, herbal medicines and products bought on the internet since these present further problems to understanding the effects of drugs. A specific example, not included in the table of sports drug history in Bird et al, relates to nandrolone and its detection. In 1998 to 2002 some athletes were found to have results indicative of abuse of nandrolone (19-nortestosterone). The cut-off concentration of nandrolone metabolites in urine was lowered so that nandrolone abuse could be detected for longer. Unfortunately, it was found that nutritional supplements were contaminated with low levels of nandrolone, sufficient to raise the concentrations of the metabolites into to the range encountered some time after nandrolone deliberate abuse.

Doping laboratory tests began with gas chromatography (GC) and progressed to GC with mass spectrometry (MS) in order to enhance specificity of identification of drugs and metabolites. One aim of this was to avoid legal challenges over the interpretation of adverse findings. In order to cover the large numbers of drugs abused in sport, MS is used in selected ion monitoring mode looking for coincident responses of diagnostic fragment ions at correct GC retention for each compound (drug or metabolites). In fact the detection of metabolites is more significant because it proves passage through the body, and thus overcomes a challenge from spiking of a sample with the drug. The process however is limited to the detection of known compounds, which requires extensive pharmacodynamic studies of drug clearance. The doping world is continuously looking for compounds that might not be detected. Tetrahydrogestrinone (THG), a potential anabolic steroid, evaded detection by GC-MS for a while, partly because of instability of the molecule during analysis. The analytical procedure was modified in response, and supposedly ‘clean’ samples were retested, revealing the extent of its abuse. Radioimmunoassay has not figured largely in the drug testing programme because of questionable specificity and the need for multiplexing to detect several possible compounds. Immuno-affinity absorption has been used as a general isolation step before MS analysis. Recombinant yeast cells have been developed for receptor assays. High performance liquid chromatography (HPLC), or LC, with MS, has been adopted to reduce sample preparation times, an important factor when handling large numbers of samples (e.g. during high-profile global competitions like the Olympics). Hydrophilic interaction LC is being used for polar compounds. Tandem mass spectrometry, time-of-flight MS, high resolution/accurate mass MS and Orbitrap have all been used to detect drug abuse ² though the equipment is more expensive than would be justified in health care laboratory budgets.