Christopher J. Howe

Carbon isotope ratio (δ13C) values of urinary steroids for doping control in sport

The detection of steroids originating from synthetic precursors in relation to their chemically identical natural analogues has proven to be a significant challenge for doping control laboratories accredited by the World Anti-Doping Agency (WADA). Endogenous steroid abuse may be confirmed by utilising the atomic specificity of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) that enables the precise measurement of differences in stable isotope ratios that arise as a result of fractionation patterns inherent in the source of steroids. A comprehensive carbon isotope ratio (delta(13)C) profiling study (n=1262) of urinary ketosteroids is reported that demonstrates the inter-individual variation that can be expected from factors such as diet, ethnicity, gender and age within and between different populations (13 countries). This delta(13)C distribution is shown by principal component analysis (PCA) to provide a statistical comparison to delta(13)C values observed following administration of testosterone enanthate. A limited collection of steroid diol data (n=100; consisting of three countries) is also presented with comparison to delta(13)C values of excreted testosterone to validate criteria for WADA accredited laboratories to prove doping offences.

Within-Subject Variability and Analytic Imprecision of Insulinlike Growth Factor Axis and Collagen Markers: Implications for Clinical Diagnosis and Doping Tests

BACKGROUND The utility of insulinlike growth factor (IGF) axis and collagen markers for a growth hormone (GH) doping test in sport depends on their stability and reproducibility. We sought to determine short-term within-subject variability of these markers in a large cohort of healthy individuals. METHODS We measured IGF-I, IGF binding protein 3 (IGFBP-3), acid labile subunit (ALS), and the collagen markers N-terminal propeptide of type I procollagen (PINP), C-terminal telopeptide of type I collagen (ICTP), and N-terminal propeptide of type III procollagen (PIIINP) in serum samples obtained on multiple occasions (median 3 per participant) over a 2- to 3-week period from 1103 elite athletes (699 men, 404 women) ages 22.2 (5.2) years [mean (SD)]. We estimated between-subject and within-subject variances by mixed-effects ANOVA. RESULTS Within-subject variance accounted for 32% to 36% and 4% to 13% of the total variance in IGF markers and collagen markers, respectively. The within-subject CV ranged from 11% to 21% for the IGF axis markers and from 13% to 15% for the collagen markers. The index of individuality for the IGF axis markers was 0.66-0.76, and for the collagen markers, 0.26-0.45. For each marker, individuals with initial extreme measured values tended to regress toward the population mean in subsequent repeated measurements. We developed a Bayesian model to estimate the long-term probable value for each marker. CONCLUSIONS These results indicate that in healthy individuals the within-subject variability was greater for IGF-I than for the collagen markers, and that where a single measurement is available, it is possible to estimate the long-term probable value of each of the markers by applying the Bayesian approach. Such an application can increase the reliability and decrease the cost of detecting GH doping.

Pharmacodynamics of growth hormone abuse biomarkers and the influence of gender and testosterone: a randomized double-blind placebo-controlled study in young recreational athletes.

CONTEXT IGF axis proteins and collagen peptides are promising markers of GH abuse. OBJECTIVE Our objective was to investigate whether responses of serum IGF axis and collagen markers to GH differ between men and women, and are influenced by testosterone (T). DESIGN This was a randomized, double-blind, placebo-controlled study of 8-wk treatment followed by 6-wk washout. SETTING The study was performed at a clinical research facility. PARTICIPANTS A total of 96 recreationally trained healthy athletes (63 men, 33 women), aged 18-40 yr, were studied. INTERVENTION All subjects received GH (2 mg/d sc) or placebo for 8 wk; men also received T (250 mg/wk im) or placebo for 5 wk. MAIN OUTCOME MEASURES Serum IGF axis proteins (IGF-I, IGF binding protein-3, and acid labile subunit) and collagen peptides (N-terminal propeptide of type I procollagen, C-terminal telopeptide of type I collagen, and N-terminal propeptide of type III procollagen) were measured. RESULTS GH induced significant increases in IGF axis and collagen markers that were greater in men than women (P < 0.001). Of the IGF axis markers, IGF-I showed the greatest increase. The relative incremental responses of the collagen markers in general were greater than the IGF markers, especially for PIIINP. The collagen markers increased and decreased more slowly with most remaining elevated (P < 0.01) after 6 wk, in comparison to IGF markers, which returned to baseline within 1 wk. Addition of T to GH amplified the response of PIIINP by more than 1.5-fold but did not affect any other marker. T alone did not affect IGF axis markers but modestly increased collagen markers. CONCLUSIONS These markers of GH abuse are less responsive in women. The increases in collagen markers have a different time course to the IGF markers and extend the window of detection in both sexes. The response of PIIINP is increased by coadministration of T.

Strategies for rhepo Detection in Sport

This article examines available strategies for the detection of recombinant erythropoietin (rhEPO) abuse in sport. RhEPO was quickly recognized as an effective but hazardous performance-enhancing agent. In the absence of a valid procedure to detect rhEPO doping, at-competition health checks were introduced, which excluded athletes from competition when their hemoglobin or hematocrit values exceeded an arbitrary limit. This limited the danger to athletes, but did nothing to eliminate the use of rhEPO.Through the last decade, both direct and indirect methods for detecting rhEPO were investigated. No single indirect marker was found that satisfactorily demonstrated rhEPO use. A combination of blood and urine tests together formed the procedure and strategy approved by the International Olympic Committee (IOC) for detecting rhEPO use at the Sydney Olympics.However strategies for testing for EPO are as important as the developed laboratory analytical procedures. The use of extensive out-of-competition testing and analysis within the IOC accredited laboratory system is critical to any testing program. At-competition blood tests have merit as true health checks and will also be needed to detect acutely useful agents such as hemoglobin-based oxygen carriers. However the persistence of the “health check” rationale for on-site at-competition rhEPO testing has led to much wasted testing effort, as rhEPO use by athletes will rarely occur near to or at the time of the competition for fear of detection. Thus, direct testing methods (such as the rhEPO urine test) especially will fail due to the completed metabolism and elimination of administered rhEPO before the test, unless the international sporting federations use the information gathered to assist in targeted out-of-competition testing. This article discusses the limitations of testing at competition and proposed strategies for dealing with various phases of EPO doping in detail, concluding that no one single currently used strategy will detect all users of rhEPO.The development of strategies to diagnose rhEPO abuse may serve as a model to detect other biological agents.

Screening for testosterone abuse in male athletes using the measurement of urinary LH, a revision of the paradigm.

The primary screening method for the detection of doping by athletes using synthetic versions of endogenous steroids such as testosterone relies on measurement of the ratio of testosterone (T) to epitestosterone (E) in urine. In 2005 the World Anti-Doping Agency (WADA) lowered the T/E value at which samples undergo further investigation from six to four. This has resulted in a large increase in the number of athletes with naturally elevated T/E ratios undergoing investigation without a corresponding increase in the number of proven doping offences involving testosterone.Our objective was to develop a new simple screening protocol that can, with high probability, not only distinguish athletes whose natural T/E values exceed four from those whose T/E values have been elevated by testosterone doping but also detect those athletes with naturally low T/E values that do not exceed four despite being administered testosterone.Testosterone (250 mg Sustanon) was administered weekly to a group of 47 young adult males for five weeks in a double-blind placebo controlled study and urine samples collected. The samples were analysed for steroid concentrations using GC/MS and for luteinizing hormone (LH) by immunoassay.The elevation of T/E that occurred in all subjects was accompanied by a significant reduction in urinary LH concentrations to levels that are rare in normal subjects.The appropriate measurement of urinary LH, with the measurement of T/E values, can markedly improve the efficiency of detection of doping with testosterone by male athletes, particularly those who have low natural T/E ratios.

Development of criteria for the detection of adrenosterone administration by gas chromatography‐mass spectrometry and gas chromatography‐combustion‐isotope ratio mass spectrometry for doping control

Adrenosterone (androst-4-ene-3,11,17-trione, 11-oxoandrostenedione) is an endogenous steroid hormone that has been promoted as a dietary supplement capable of reducing body fat and increasing muscle mass. It is proposed that adrenosterone may function as an inhibitor of the 11beta-hydroxysteroid dehydrogenase type 1 enzyme (11beta-HSD1), which is primarily responsible for reactivation of cortisol from cortisone. The urinary metabolism of adrenosterone was investigated, after a single oral administration in two male subjects, by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Substantially increased excretion of 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, 11-oxoandrosterone and 11-oxoetiocholanolone was observed. Minor metabolites such as 3alpha,17beta-dihydroxy-5beta-androstan-11-one, 3alpha-hydroxyandrost-4-ene-11,17-dione and 3alpha,11beta-dihydroxyandrost-4-en-17-one were also identified. The exogenous origin of the most abundant adrenosterone metabolites was confirmed by GC-C-IRMS according to World Anti-Doping Agency criteria. Through analysis of a reference population data set obtained from urine samples provided by elite athlete volunteers (n = 85), GC-MS doping control screening criteria are proposed: 11beta-hydroxyandrosterone concentration greater than 10 000 ng/mL (specific gravity adjusted to 1.020) or 11beta-hydroxyandrosterone/11beta-hydroxyetiocholanolone ratio greater than 20.Urine samples fulfilling these screening criteria may be subjected to GC-C-IRMS analysis for confirmation of adrenosterone administration.

Erythropoietin administration does not influence the GH--IGF axis or makers of bone turnover in recreational athletes.

Objective  Measurement of biochemical markers of the IGF‐system and of collagen turnover is a potential approach to detect GH abuse in sport. These markers are increased in patients on dialysis treated with recombinant human erythropoietin (r‐HuEPO), mimicking the effects of GH. The aim was to determine whether r‐HuEPO induces similar effects on the IGF‐system and collagen turnover in healthy athletes.