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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.

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.

Context: GH-responsive markers of the IGF system and of collagen turnover hold promise as the basis of a GH doping test., Objective: The purpose of this study was to determine the influence of age, gender, body mass index (BMI), ethnicity, and sporting type on GH-responsive serum markers in a large cohort of elite athletes from different ethnic backgrounds., Design: The study was designed as a cross-sectional study., Participants: A total of 1103 elite athletes (699 males, 404 females), aged 22.2 +/- 5.2 yr, from 12 countries and 10 major sporting categories participated in this study., Main Outcome Measures: Serum IGF-I, IGF binding protein-3 (IGFBP-3), acid labile subunit (ALS), and collagen markers [N-terminal propeptide of type I procollagen (PINP), C-terminal telopeptide of type I collagen (ICTP), N-terminal propeptide of type III procollagen (PIIINP)] were measured., Results: There was a significant negative correlation (r = -0.14 to -0.58, P < 0.0005) between age and each of the GH-responsive markers. Serum IGF-I, IGFBP-3, and ALS were all lower (P < 0.05), whereas the collagen markers PINP, ICTP, and PIIINP were higher (P < 0.05) in men than in women. Multiple regression analysis indicated that age, gender, BMI, and ethnicity accounted for 23-54% of total between-subject variability of the markers. Age and gender cumulatively accounted for 91% of the attributable variation of IGF-I and more than 80% for PINP, ICTP, and PIIINP. Gender exerted the greatest effect on ALS (48%), and BMI accounted for less than 12% attributable variation for all markers. The influence of ethnicity was greatest for IGFBP-3 and ALS; however, for the other markers, it accounted for less than 6% attributable variation. Analysis of 995 athletes indicated that sporting type contributed 5-19% of attributable variation., Conclusions: Age and gender were major determinants of variability of GH-responsive markers except for IGFBP-3 and ALS. Ethnicity is unlikely to confound the validity of a GH doping test based on IGF-I and these collagen markers.

Context: Management of male infertility and/or androgen deficiency requires accurate hormonal measurements with valid reference intervals., Objective: The objective of this study was to develop a valid reference panel of blood samples from healthy eugonadal young men with verified normal reproductive function and to use this panel to evaluate the performance of seven fully automated, commercial multiplex immunoassay platforms used to measure serum total testosterone (T), LH, and FSH., Design: This was an observational study of consistency among seven different automated immunoassays for each of total T, LH, and FSH. Each method was implemented in two laboratories, with each repeating the analysis of the full reference panel samples twice. Serum T concentrations were also measured by gas chromatography/mass spectrometry (GC/MS), and serum inhibin B levels were determined by an ELISA., Setting: The study was performed at commercial, high-volume, clinical pathology laboratories., Participants: From 147 men screened, sera from 124 healthy, reproductively normal men (age, 21-35 yr) with normal sperm output were used as a reference panel. All laboratories selected for elite performance in the national immunoassay quality assurance program agreed to participate., Main Outcome Measure(s): For each of the 868 assays, descriptive statistics were calculated in the natural and log-transformed scales and were analyzed by nested, repeated measures ANOVA after log transformation. Reference intervals, defined as 95% confidence limits, were calculated using arithmetic (natural scale), geometric (log scale) and nonparametric methods., Results: Descriptive statistics and reference intervals for serum T, LH, and FSH differed widely and significantly between methods, but variation between laboratories for the same assay was negligible. All T methods showed significant differences in regression slope and intercept in deviance plots as well as in estimated reference ranges compared with the independent GC/MS reference method. Although similar between-method differences existed for gonadotropin assays, the smaller quantitative discrepancies allowed assignment of consensus reference intervals for serum FSH (1.3-8.4 IU/liter) and LH (1.6-8.0 IU/liter), although these differed from manufacturers' currently quoted expected values., Conclusions: Using a reference panel of sera from healthy eugonadal young men with verified normal reproductive function, major differences exist between commercial T immunoassays as well as divergence from the GC/MS standard. This impairs their clinical diagnostic utility and requires substantial improvements in automated T immunoassay technologies or a switch to GC/MS methods. Gonadotropin assays showed less variability, but current high-throughput immunoassays remain suboptimal to confirm accurate diagnosis of azoospermia or androgen deficiency.

Blood substitutes based on hemoglobin or hemoglobin-based oxygen carriers (HBOCs) are oxygen-carrying therapeutic agents developed for use in operations and emergencies in place of donated blood. Increased oxygen-carrying capacity through the use of blood substitutes could help elite athletes to lengthen endurance capacity and improve their performance. As blood substitutes become more readily available, it is essential that a qualitative detection method for their abuse in sport is available. Ideally, such a method would be simple and inexpensive. This study investigates methods that could be used as screening procedures to easily detect HBOCs in plasma and develops tests that can unequivocally confirm their presence. The investigation into the screening method indicates that the direct visual screening of plasma discoloration is the most appropriate with detection limits of less than 1% HBOC in plasma. Two methods are shown to confirm the presence of exogenous hemoglobin in plasma samples, size-exclusion chromatography with photodiode array detection and high-performance liquid chromatography analysis of enzymatic digests with detection by electrospray mass spectrometry. This work emphasizes the need for cooperation between drug developers and sports testing laboratories to ensure that methods for the detection of putative doping agents are available prior to product release.

The use of gas chromatography (GC)-combustion (C)-isotope ratio mass spectrometry (IRMS) demonstrates that a single oral administration of dehydroepiandrosterone (DHEA, 100 mg) to a male subject significantly lowers the 13C content of etiocholanolone (Et) and androsterone (A) in the subject's urine. The difference in carbon isotope ratio (d13C per thousand) values between Et and A increases from 1.6 per thousand at the time of administration to 5.1 per thousand at 26 h post-administration, indicating preferential metabolism of administered DHEA to form Et in relation to A. Multiple oral administrations of DHEA to a male subject reveals lower d13C values during the excretion period of Et (-31.7 per thousand to -34.6 per thousand) and A (-31.4 per thousand to -33.0 per thousand) to that of the d13C value of the administered DHEA (-31.3 per thousand). Reference distributions of d13C Et and d13C A constructed from normal athlete populations within Australia and New Zealand show a small natural discrimination against 13C in the formation of Et relative to A (mean=0.3 per thousand, n=167, p=0.007). Amplified differences between d13C Et and d13C A, and in vivo 13C depletion measured by GC-C-IRMS are shown to be potentially useful for doping control.

Tetrahydrogestrinone (THG) was recently identified as a novel steroid used illicitly to improve athletic performance. Although its structure is closely related to gestrinone, a 19-nor progestin, and resembles that of trenbolone, THG was never marketed, so information on its hormonal properties is not known. In this study, we demonstrate that THG is a highly potent androgen and progestin in a yeast-based in vitro bioassay system expressing human androgen and progesterone receptors. It has no estrogenic activity and no antagonism for any of the three steroid receptor classes.