The effects of recombinant human insulin-like growth factor-1/insulin-like growth factor binding protein-3 administration on lipid and carbohydrate metabolism in recreational athletes.

OBJECTIVE: Previous studies suggested that recombinant human IGF-1 (rhIGF-1) administration affects carbohydrate and lipid metabolism in healthy people and in people with diabetes. This study aimed to determine the effects of rhIGF-1/rhIGF binding protein-3 (rhIGFBP-3) administration on glucose homeostasis and lipid metabolism in healthy recreational athletes. DESIGN AND SETTING: Randomized, double-blind, placebo-controlled rhIGF-1/rhIGFBP-3 administration study at Southampton General Hospital, UK. PARTICIPANTS: 56 recreational athletes (30 men, 26 women). METHODS: Participants were randomly assigned to receive placebo, low-dose rhIGF-1/rhIGFBP-3 (30 mg/day) or high-dose rhIGF-1/rhIGFBP-3 (60 mg/day) for 28 days. The following variables were measured before and immediately after the treatment period: fasting lipids, glucose, insulin, C-peptide and glycated haemoglobin. The homeostatic model assessment (HOMA-IR) was used to estimate insulin sensitivity and indirect calorimetry to assess substrate oxidation rates. The general linear model approach was used to compare treatment group changes with the placebo group. RESULTS: Compared with the placebo group, there was a significant reduction in fasting triglycerides in participants treated with high-dose rhIGF-1/rhIGFBP-3 (p = .030), but not in the low-dose group (p = .390). In women, but not in men, there were significant increases in total cholesterol (p = .003), HDL cholesterol (p = .001) and LDL cholesterol (p = .008). These lipid changes were associated with reduced fasting insulin (p = .010), C-peptide (p = .001) and HOMA-IR (p = .018) in women and reduced C-peptide (p = .046) in men. CONCLUSIONS: rhIGF-1/rhIGFBP-3 administration for 28 days reduced insulin concentration, improved insulin sensitivity and had significant effects on lipid profile including decreased fasting triglycerides.

Comparison of normal distribution-based and nonparametric decision limits on the GH-2000 score for detecting growth hormone misuse (doping) in sport.

This paper is motivated by the GH-2000 biomarker test, though the discussion is applicable to other diagnostic tests. The GH-2000 biomarker test has been developed as a powerful technique to detect growth hormone misuse by athletes, based on the GH-2000 score. Decision limits on the GH-2000 score have been developed and incorporated into the guidelines of the World Anti-Doping Agency (WADA). These decision limits are constructed, however, under the assumption that the GH-2000 score follows a normal distribution. As it is difficult to affirm the normality of a distribution based on a finite sample, nonparametric decision limits, readily available in the statistical literature, are viable alternatives. In this paper, we compare the normal distribution-based and nonparametric decision limits. We show that the decision limit based on the normal distribution may deviate significantly from the nominal confidence level 1-α or nominal FPR γ when the distribution of the GH-2000 score departs only slightly from the normal distribution. While a nonparametric decision limit does not assume any specific distribution of the GH-2000 score and always guarantees the nominal confidence level and FPR, it requires a much larger sample size than the normal distribution-based decision limit. Due to the stringent FPR of the GH-2000 biomarker test used by WADA, the sample sizes currently available are much too small, and it will take many years of testing to have the minimum sample size required, in order to use the nonparametric decision limits. Large sample theory about the normal distribution-based and nonparametric decision limits is also developed in this paper to help understanding their behaviours when the sample size is large.

Exact statistical calculation of the uncertainty term in the decision limits of the GH-2000 score for growth hormone misuse (doping) detection.

The GH-2000 score has been developed as a powerful and unique technique for the detection of growth hormone misuse by sportsmen and women. The score depends upon the measurement of two growth hormone sensitive markers, insulin-like growth factor-I and the amino-terminal pro-peptide of type III collagen. It also includes a term to adjust for the age of the athlete. Decision limits for the GH-2000 score have been developed and are incorporated into the guidelines of the World Anti-Doping Agency. These decision limits are derived by setting a 1 in 10,000 false-positive rate rule. As these decision limits are estimated from samples of GH-2000 scores, they carry uncertainty. In previous work, this uncertainty has been addressed by establishing an upper 95% confidence interval for the true decision limits based on a normal approximation which has been shown to be appropriate if sample sizes are large (such as 1000 and above). Here, we show that these approximations, whether reasonable or not, can be entirely avoided by developing an upper 95% confidence interval for the true decision limits using an approach based upon the t-distribution. While there are considerable differences for smaller sample sizes, these become negligible when the sample size is large such as 1000 and above.

A correction to the age-adjustment of the GH-2000 score used in the detection of growth hormone misuse.

OBJECTIVE: The GH-2000 biomarker test has been introduced by the World Anti-Doping Agency as a method of detecting growth hormone misuse in professional sport. The test involves the measurement insulin-like growth factor-I and the amino-terminal pro-peptide of type III collagen (P-III-NP) which increase in a dose-dependent manner in response to GH. These measurements are combined in sex specific formulae that include an age adjustment. The original age adjustment overcorrects the effect of age in male athletes and could potentially place older men at a disadvantage. The purpose of this note is to investigate the performance of a previously suggested correction term in two new and larger data sets. RESULTS: The GH-2000 score was calculated for 7307 samples obtained from 15 accredited WADA laboratories in 2017 and 3916 samples measured at Drug Control Centre, King's College London, UK between 2013 and 2017. The GH-2000 scores were investigated for positive age effects using standard regression modelling. As previously, all analyses confirmed a positive age effect. Applying the earlier suggested correction term of 0.032 × age showed a significant over-correction leading to a negative association of the GH-2000 score with age. We now suggest a smaller age correction of 0.020 × age, which corresponds to the smallest effect found in the earlier studies.

Why do endocrine profiles in elite athletes differ between sports?

BACKGROUND: Endocrine profiles have been measured on blood samples obtained immediately post-competition from 693 elite athletes from 15 Olympic Sports competing at National or International level; four were subsequently excluded leaving 689 for the current analysis. METHODS: Body composition was measured by bioimpedance in a sub-set of 234 (146 men and 88 women) and from these data a regression model was constructed that enabled 'estimated' lean body mass and fat mass to be calculated on all athletes. One way ANOVA was used to assess the differences in body composition and endocrine profiles between the sports and binary logistical regression to ascertain the characteristic of a given sport compared to the others. RESULTS: The results confirmed many suppositions such as basketball players being tall, weightlifters short and cross-country skiers light. The hormone profiles were more surprising with remarkably low testosterone and free T3 (tri-iodothyronine) in male powerlifters and high oestradiol, SHBG (sex hormone binding globulin) and prolactin in male track and field athletes. Low testosterone concentrations were seen 25.4% of male elite competitors in 12 of the 15 sports and high testosterone concentrations in 4.8% of female elite athletes in 3 of the 8 sports tested. Interpretation of the results is more difficult; some of the differences between sports are at least partially due to differences in age of the athletes but the apparent differences between sports remain significant after adjusting for age. The prevalence of 'hyperandrogenism' (as defined by the IAAF (International Association of Athletics Federations) and IOC (International Olympic Committee)) amongst this cohort of 231 elite female athletes was the highest so far recorded and the very high prevalence of 'hypoandrogenism' in elite male athletes a new finding. CONCLUSIONS: It is unclear whether the differences in hormone profiles between sports is a reason why they become elite athletes in that sport or is a consequence of the arduous processes involved. For components of body composition we know that most have a major genetic component and this may well be true for endocrine profiles.

Determination and regulation of body composition in elite athletes.

In 2011, the International Association of Athletics Federations (IAAF) and IOC introduced a 'hyperandrogenism' rule that excluded women with a serum testosterone >10 nmol/L from participating in elite sport. This rule was based on the false premise that the greater lean body mass in men was a consequence of their higher serum testosterone. This rule did not have scientific backing and the Court of Arbitration for Sport subsequently rescinded the rule following an appeal from an Indian athlete barred from the Commonwealth Games. This review covers the scientific knowledge about the development and regulation of body composition in humans but also considers the lessons learnt from evolution and breeding in animals. The importance of heredity has been documented in family and twin studies. The roles of growth hormone and sex steroids are reviewed. The Androgen Insensitivity Syndrome (AIS) is considered as a model of the role of testosterone in development of body composition and also as evidence of the importance of other factors carried on the Y-chromosome that are of prime importance but have been systematically ignored. Finally the key factors determining body composition are considered and placed in a suggested order of importance.

Novel markers to detect recombinant human insulin-like growth factor-I (rhIGF-I)/rhIGF binding protein-3 (rhIGFBP-3) misuse in athletes.

Insulin-like growth factor-I (IGF-I) is abused by elite athletes for its metabolic and anabolic effects. We have previously shown that it is possible to detect IGF-I misuse by measuring serum IGF-I and procollagen type III amino-terminal propeptide (P-III-NP) but a pilot study suggested measuring IGF-II, IGF binding protein-2 (IGFBP-2) and acid-labile subunit (ALS) may improve the detection of IGF-I administration. The aim of the study was to assess this in a randomized controlled trial. Twenty-six female and 30 male recreational athletes were randomized to 28 days' treatment with placebo or recombinant human (rh)IGF-I/rhIGF binding protein-3 (IGFBP-3) complex (30 mg/day or 60 mg/day), followed by 56 days' washout. IGF-II, IGFBP-2 and ALS (women only) were measured using commercial immunoassays. IGFBP-2 increased and IGF-II decreased in response to both low and high dose rhIGF-I/rhIGFBP-3 in both women and men while ALS decreased in women in response to high dose rhIGF-I/rhIGFBP-3. Two days after discontinuing treatment, significant differences remained between the three treatment groups in IGFBP-2 and IGF-II, but not ALS. Thereafter there were no significant differences between the three treatment groups in any of the markers. Combining IGF-I with IGF-II and/or IGFBP-2 improved the performance of the test to detect rhIGF-I/rhIGFBP-3 administration in both women and men. Copyright © 2016 John Wiley & Sons, Ltd.

Statistical methodology for age-adjustment of the GH-2000 score detecting growth hormone misuse.

BACKGROUND: The GH-2000 score has been developed as a powerful and unique technique for the detection of growth hormone misuse by sportsmen and women. The score depends upon the measurement of two growth hormone (GH) sensitive markers, insulin-like growth factor-I (IGF-I) and the amino-terminal pro-peptide of type III collagen (P-III-NP). With the collection and establishment of an increasingly large database it has become apparent that the score shows a positive age effect in the male athlete population, which could potentially place older male athletes at a disadvantage. METHODS: We have used results from residual analysis of the general linear model to show that the residual of the GH-2000 score when regressed on the mean-age centred age is an appropriate way to proceed to correct this bias. As six GH-2000 scores are possible depending on the assays used for determining IGF-I and P-III-NP, methodology had to be explored for including six different age effects into a unique residual. Meta-analytic techniques have been utilized to find a summary age effect. RESULTS: The age-adjusted GH-2000 score, a form of residual, has similar mean and variance as the original GH-2000 score and, hence, the developed decision limits show negligible change when compared to the decision limits based on the original score. We also show that any further scale-transformation will not change the adjusted score. Hence the suggested adjustment is optimal for the given data. The summary age effect is homogeneous across the six scores, and so the generic adjustment of the GH-2000 score formula is justified. CONCLUSIONS: A final revised GH-2000 score formula is provided which is independent of the age of the athlete under consideration.

The Effects of Recombinant Human Insulin-Like Growth Factor-I/Insulin-Like Growth Factor Binding Protein-3 Administration on Body Composition and Physical Fitness in Recreational Athletes.

CONTEXT: IGF-I is thought to mediate many of the anabolic actions of GH, and there are anecdotal reports that IGF-I is misused by elite athletes. There is no published evidence regarding the effects of IGF-I administration on athletic performance. OBJECTIVE: The objective of the study was to investigate the effects of IGF-I administration on body composition and physical fitness in recreational athletes. DESIGN AND SETTING: This was a randomized, double-blind, placebo-controlled recombinant human (rh) IGF-I/rhIGF binding protein (IGFBP)-3 administration study at Southampton General Hospital (Southampton, United Kingdom). PARTICIPANTS: Fifty-six recreational athletes (30 men, 26 women) participated in the study. INTERVENTION: Participants were randomly assigned to receive placebo, low-dose rhIGF-I/rhIGFBP-3 (30 mg/d), or high dose rhIGF-I/rhIGFBP-3 (60 mg/d) for 28 days. Body composition (assessed by dual energy x-ray absorptiometry) and cardiorespiratory fitness (assessed by incremental treadmill test) were measured before and immediately after treatment. Within-individual changes after treatment were analyzed using paired t tests. RESULTS: There were no significant changes in body fat mass or lean body mass in women or men after the administration of the rhIGF-I/rhIGFBP-3 complex. There was a significant increase in maximal oxygen consumption (VO2 max) after treatment. When women and men and low- and high-dose treatment groups were combined, mean VO2 max increased by approximately 7% (P = .001). No significant change in VO2 max was observed in the placebo group. CONCLUSIONS: rhIGF-I/rhIGFBP-3 administration for 28 days improves aerobic performance in recreational athletes, but there are no effects on body composition.

The development of decision limits for the GH-2000 detection methodology using additional insulin-like growth factor-I and amino-terminal pro-peptide of type III collagen assays.

The GH-2000 and GH-2004 projects have developed a method for detecting GH misuse based on measuring insulin-like growth factor-I (IGF-I) and the amino-terminal pro-peptide of type III collagen (P-III-NP). The objectives were to analyze more samples from elite athletes to improve the reliability of the decision limit estimates, to evaluate whether the existing decision limits needed revision, and to validate further non-radioisotopic assays for these markers. The study included 998 male and 931 female elite athletes. Blood samples were collected according to World Anti-Doping Agency (WADA) guidelines at various sporting events including the 2011 International Association of Athletics Federations (IAAF) World Athletics Championships in Daegu, South Korea. IGF-I was measured by the Immunotech A15729 IGF-I IRMA, the Immunodiagnostic Systems iSYS IGF-I assay and a recently developed mass spectrometry (LC-MS/MS) method. P-III-NP was measured by the Cisbio RIA-gnost P-III-P, Orion UniQ™ PIIINP RIA and Siemens ADVIA Centaur P-III-NP assays. The GH-2000 score decision limits were developed using existing statistical techniques. Decision limits were determined using a specificity of 99.99% and an allowance for uncertainty because of the finite sample size. The revised Immunotech IGF-I - Orion P-III-NP assay combination decision limit did not change significantly following the addition of the new samples. The new decision limits are applied to currently available non-radioisotopic assays to measure IGF-I and P-III-NP in elite athletes, which should allow wider flexibility to implement the GH-2000 marker test for GH misuse while providing some resilience against manufacturer withdrawal or change of assays.

Biochemical markers of insulin-like growth factor-I misuse in athletes: the response of serum IGF-I, procollagen type III amino-terminal propeptide, and the GH-2000 score to the administration of rhIGF-I/rhIGF binding protein-3 complex.

CONTEXT: The GH-2000 and GH-2004 research groups developed a method for detecting GH misuse in athletes based on the measurement of serum IGF-I and procollagen type III amino-terminal propeptide (P-III-NP). There are reports that IGF-I is also misused by athletes, but currently there is no internationally recognized test designed to detect recombinant human IGF-I misuse. OBJECTIVE: The objective of the study was to examine the response of serum IGF-I, P-III-NP, and the GH-2000 score to recombinant human (rh) IGF-I/rhIGF binding protein-3 (IGFBP-3) administration in recreational athletes. DESIGN AND SETTING: This was a randomized, double-blind, placebo-controlled rhIGF-I/rhIGFBP-3 administration study at Southampton General Hospital (Southampton, United Kingdom). PARTICIPANTS: Fifty-six recreational athletes (26 women, 30 men) participated in the study. INTERVENTION: Participants were randomized to treatment with low-dose (30 mg/d) or high-dose (60 mg/d) rhIGF-I/rhIGFBP-3 complex or placebo for 28 days. Blood was collected throughout the drug administration and washout periods. Serum IGF-I and P-III-NP were measured using commercial immunoassays and GH-2000 scores were calculated. RESULTS: IGF-I, P-III-NP, and the GH-2000 score rose in response to both low- and high-dose rhIGF-I/rhIGFBP-3 administration. The relative maximum response of IGF-I (approximately 4-fold increase in women and men) was greater than that of P-III-NP (40%-50% increase in women, 35%-50% increase in men). The GH-2000 formulae, which incorporate IGF-I and P-III-NP results, detected up to 61% of women and 80% of men in the rhIGF-I/rhIGFBP-3 groups but, using IGF-I concentrations alone, the sensitivity increased to 94% in both women and men during the administration period. CONCLUSIONS: The rise in P-III-NP after rhIGF-I/rhIGFBP-3 administration is small compared with that after rhGH administration. Although rhIGF-I/rhIGFBP-3 administration can be detected using the GH-2000 score method, a test based on serum IGF-I alone provides better sensitivity.

Biochemical markers of recombinant human insulin-like growth factor-I (rhIGF-I)/rhIGF binding protein-3 (rhIGFBP-3) misuse in athletes.

Insulin-like growth factor-I (IGF-I) is reportedly misused by elite athletes, either alone or with growth hormone (GH). The GH-2000 and GH-2004 research groups previously developed a method for detecting GH misuse based on the GH-sensitive markers IGF-I and procollagen type III amino-terminal propeptide (P-III-NP). Both markers increase in response to rhIGF-I/rhIGF binding protein-3 (rhIGFBP-3) administration in recreational athletes. The aim of this pilot study was to assess the effect of rhIGF-I/rhIGFBP-3 administration on other serum markers of the GH-IGF axis and on other bone and collagen markers. Twenty-six female and 30 male recreational athletes were randomized to 28 days' treatment with placebo or rhIGF-I/rhIGFBP-3 complex, followed by 56 days' washout. GH-IGF axis markers (IGFBP-2, IGFBP-3, acid-labile subunit (ALS) and IGF-II) and bone and collagen markers (procollagen type I carboxy-terminal propeptide (PICP), type I collagen cross-linked carboxy-terminal telopeptide (ICTP) and osteocalcin) were measured using commercial immunoassays. In women in the high dose treatment group, mean IGF-II decreased by 53% (P=0.0028) on Day 21. Mean IGFBP-2 increased by 119% (P=0.0039) and mean ALS decreased by 40% (P=0.0022) on Day 21. There were no significant changes in IGFBP-3, osteocalcin, ICTP or PICP. In men in the high dose group, mean IGF-II decreased by 51% on Day 21 (P<0.0001). Mean IGFBP-2 increased by 125% on Day 21 (P=0.0003). There were no significant changes in IGFBP-3, ALS, osteocalcin, ICTP or PICP. Serum IGFBP-2 and IGF-II may be useful markers of rhIGF-I/rhIGFBP-3 administration in both women and men while ALS may also be a useful marker in women; these markers are now undergoing further evaluation.

Insulin-like growth factor-I (IGF-I) misuse in athletes and potential methods for detection.

To athletes, insulin-like growth factor-I (IGF-I) is an attractive performance-enhancing drug, particularly as an alternative to growth hormone (GH) because IGF-I mediates many of the anabolic actions of GH. IGF-I has beneficial effects on muscle protein synthesis and glycogen storage that could enhance performance in several sporting disciplines. Recombinant human IGF-I (rhIGF-I) is used in clinical practice, but a variety of IGF-I compounds and IGF-I analogues are also advertised on the internet and many have been available on the black market for several years. Although methods for detecting GH misuse are now well established and there have been several cases in which athletes have tested positive for GH, no test is yet in place for detecting IGF-I misuse. The GH-2004 research group has been investigating methods for detection of IGF-I misuse and a test is being developed on the basis of the principles of the successful GH-2000 marker method, in which markers from the IGF axis and markers of collagen and bone turnover are used to detect GH misuse. Commercial immunoassays for these markers have been validated for anti-doping purposes but new methods, including IGF-I measurement by use of mass spectrometry, should improve the performance of the tests and help in the detection of athletes who are doping with these peptide hormones.