Two main approaches have been investigated to detect GH abuse; the first is based on the detection of different pituitary GH isoforms, whereas the second relies on measurement of GH-dependent proteins. Both methods use immunologic assays and are therefore subject to the WADA requirement that 2 separate assays detecting different epitopes are needed to verify the presence of the isoform or marker (5).
The analysis of GH isoforms was originally termed the "direct method", but it is now more accurately referred to as the "isoform assay method". Endogenous GH exists in several forms: the 22-kDa isoform is the most abundant, constituting 75% of the circulating GH, and others forms, collectively termed "non–22-kDa", include the 20- and 17-kDa isoforms and many fragments of isoforms.
Recombinant GH contains only the 22-kDa isoform, and exogenous rhGH administration leads to a marked decrease in the endogenous pituitary-derived non–22-kDa isoforms by negative feedback mechanisms. Hence, a high ratio of 22- to non–22-kDa has been proposed as a mechanism of detecting exogenous GH usage (Fig. 1) (21). Age, sex, physiologic stimulus, and pathologic state do not affect the relative proportions of GH isoforms, but it is unclear whether ethnicity could affect the isoform ratio (22). Exercise causes an increase in the 22-kDa isoform; therefore, postrace concentrations of this isoform relative to the other isoforms may lower the test’s sensitivity (23).
GH isoforms have short half-lives; therefore, the window of opportunity for detection is short, up to (at the very most) 36 h (21). Spontaneous GH secretion returns to baseline values 48 h after the last dose of GH treatment (24).
The isoform method cannot detect pituitary-derived GH doping (pituitary-derived GH from both animals and humans is still commercially available) or the abuse of GH secretagogues or IGF-I.
gh isoform assays
RIA and IRMA will detect circulating whole isoforms, whereas the immunofunctional assay methodology (IFA) has the advantage of detecting only biologically active GH isoforms. The values obtained from the IFA methods are 26.8% lower than those obtained from conventional IRMAs, reflecting the proportion of GH that is biologically active. However, the use of immunoassay techniques in forensic toxicology remains controversial despite widespread acceptance in laboratory scientific practice (4).
IFA is an ELISA method that relies on the two GH-binding sites of the transmembrane GH receptor: one binding GH and the other binding a radiolabeled monoclonal antibody (25). The solution is flooded with GH-binding protein (GHBP) to bind any remaining unoccupied site 1 epitopes. Nonfunctional isoforms do not bind the GHBP or induce dimerization, which is a critical step in bioactivity, and are thus not detected by the assay. The assay is technically easy to perform, has high capacity, and allows radiolabeling and metric or colorimetric quantification; however, the structural format of the molecule may be such that the monoclonal antibody site is unavailable for the radiolabel to attach, leading to underestimation of the true concentration. Sites already bound by GHBP will not be detected, but in vivo these molecules are too large to pass through the capillary membrane and achieve biological activity (26). The reported interassay and intraassay CVs are 10.3% and 7.3%, respectively, with no interference from endogenous GHBP (25).
The presence of fragments of isoforms that may contain binding sites for antibodies can lead to overestimation of the concentrations of whole and functional isoforms present. For example, the 22-kDa exclusion assays demonstrate incomplete exclusion of 22-kDa isoforms at high concentrations (27).
The anabolic actions of GH lead to generation of several proteins, and the serum concentrations or ratios of these proteins can be used as a means of detecting exogenous GH. This method of GH detection is known as the "marker method". The measurement of multiple markers in conjunction with specific equations, "discriminant functions", can be used to detect GH abuse with enhanced sensitivity and specificity compared with single-marker analysis. Two groups of potential markers were identified by the GH-2000 research team: one group includes members of the IGF-IGFBP axis, and the other includes markers of bone and collagen turnover and mineralization (17).
IGF-I is an ideal candidate marker because it has little diurnal or day-to-day variation (22), increases 1.3- to 2.3-fold in a uniform dose-dependent fashion after GH administration (28), and has low basal scatter (29) and minimal changes with exercise. Ninety-five percent of circulating IGF-I is bound to binding proteins (IGFBP-1 to -6), predominantly IGFBP-3, which modulate its actions and bioavailability (22)(30). IGFBP-3 increases with GH administration but has a less uniform and dose-dependent curve than IGF-I (28). Total IGF-I is used because free IGF-I is less responsive to GH (31).
Similarly, several bone and soft tissue markers change in response to GH administration. Procollagen III terminal peptide (P-III-P) is a marker of type 3 collagen formation (mainly soft tissues); exhibits little day-to-day, diurnal, or gender variation in basal concentrations; and increases in a dose-dependent fashion after GH administration (17). As a soft tissue marker, P-III-P may increase after injury and with several pathologic conditions, including fibrotic lung or liver disease or alcoholic liver disease (32). The GH-2004 team, based in Southampton, is currently evaluating the effects of different types of injury on plasma concentrations of P-III-P and other markers of GH action. C-Terminal propeptide of collagen type I (PICP) is involved in the early scaffold of collagen formation and callus formation with bone remodeling and increases in a non–dose-dependent manner after GH administration. C-Terminal cross-linked telopeptide of type I collagen (ICTP) is a marker of bone resorption and exhibits a short phase increase of 9.7% with males exhibiting an increase greater in magnitude (29).
Osteocalcin and bone alkaline phosphatase are both markers of bone mineralization (Table 1). After GH administration, osteocalcin concentrations increase in a non–dose-dependent manner, and males exhibit a greater increase than females (31). There is a small increase 60 min after exercise. Bone-specific alkaline phosphatase is posture dependent and increases slowly after GH administration (33). Leptin is derived from adipocytes in response to GH, but changes in this marker are too variable for practical use (28).
discriminant function analysis
Single-marker analysis lacks sufficient specificity to detect exogenous GH abuse; therefore, a combination of markers is used in discriminant function analysis to improve sensitivity and specificity. To generate these equations, a training set of data is applied to create the discriminant model, and a confirmatory set of data is then similarly applied to ensure that the model is applicable to the population in general and not just the sample set.
Kicman et al. (34) proposed the use of ratios of IGF-I to IGFBP-2 and IGFBP-3 to IGFBP-2, both of which were augmented at 30 h after GH administration.
The GH-2000 team reported that IGF-I and P-III-P were the simplest combination of the numerous markers studied providing the best sensitivity and specificity during GH therapy (29). Other markers of bone and collagen turnover also provided useful discrimination between placebo and treatment groups (Fig. 2).
Kniess et al. (28) reported significant increases in the concentrations of the products of the terms (IGF-I x P-III-P) and (IGF-I x IGFBP-3) in a GH administration study (Fig. 3).
The differences in time course of the markers can be exploited by mathematical modeling to improve the sensitivity of the test to detect GH abuse. Comparing the time-dependent proportionate decreases in concentrations of the markers can allow determination of the time since doping last occurred.
creation of appropriate reference intervals for gh-dependent markers
It is important to construct appropriate reference intervals for each of the proposed GH markers. Natural differences in responses of the GH-IGF axis among individuals and among differing body habitus may confer an advantage in sport and lead to differences in marker concentrations.
Exercise is a potent stimulus for GH secretion, and both acute and chronic physical activity increase the concentrations of some of the GH markers. This effect is augmented by GH administration, but the pattern of the increase is unchanged. Total, but not free, IGF-I and IGFBP-1 both increase after acute exercise (33). Acute exercise also induces a metabolic acidosis that stimulates osteoclasts and inhibits osteoblastic activity, whereas in the longer term, resistance training induces a greater increase in bone remodeling than does endurance training (35). Postcompetition concentrations of GH markers in elite athletes are different from the values in standard reference intervals (36).
An appropriate sex- and age-specific reference interval is important. Women have lower GH peaks and higher troughs and are relatively more GH resistant (10)(22). GH secretion increases through childhood and early adulthood and thereafter decreases by 14% per decade. There is also a decrease in the secretion of IGF-I and IGFBP-3 with aging (37). Athletes exhibit the same decrease in GH-dependent markers as the general population, and any differences in markers between sporting events are largely explained by differences in the ages of the competitors. Therefore, any antidoping tests for GH must take age into account. This is particularly important in adolescent competitors because during that age period, pubertal staging rather than chronologic age determines GH secretion.
The GH-2004 team based in Southampton and an Australian and Japanese Consortium are currently evaluating the possibility of ethnic differences in the GH-dependent markers. Although there are small differences in the mean concentrations of some markers, notably IGFBP-3, among ethnic groups, preliminary data suggest that these are not large enough to affect the performance of the test proposed by the GH-2000 team.
gh marker assays
Several key areas need to be clarified before introduction of the "marker" test. There have been calls for urgent standardization of calibrants and standards as well lower heterogeneity of antibodies and assay reagents for comparison (38)(39).
To improve the accuracy of the IGF-I assays, high-affinity antibodies should be used wherever possible because IGFBPs have similar affinities and compete with conventional antibodies. IGFBPs should be dissociated and separated from the IGF-I before assay, e.g., by acidification (40). Acid ethanol extraction followed by cryoprecipitation is a refinement of the size-exclusion method that has a considerably improved IGF-I recovery of 90%–95%.
The authors of a recent report comparing IGF-I assays (Nichols RIA, Mediagnost RIA, and R&D ELISA) reported variability attributable to IGF-I dissociation procedure, calibrators used, IGF-I concentrations, and antibody specificity (41). The authors reported that interlaboratory correlation was best for the ELISA for IGF-I and the Orion Diagnostics RIA for P-III-P (41).
Potential alterations in commercial assays by manufacturers could mean that a new reference interval is required for each new assay (42). There thus is a specific need for WADA to develop its own assays over which it would have complete control to ensure standardization.
The Court of Arbitration of Sport has yet to decide on an acceptable false-positive rate, but it is expected to be in the range of 1 in 10 000 tests. Current medical practice accepts as "normal" values within 2 SD from the mean on a calibration curve. By definition, this means that 5% of the population lie outside the "normal range" and would create an unacceptably high false-positive rate if applied to athletes.
Although the aim of antidoping organizations and committees is to ensure that competition is fair and free of drugs, antidoping testing has its own ethical considerations. Although largely beyond the scope of this review, it is important to ensure that testing for GH does not cause undesirable ethical and medical consequences that may outweigh the advantages for sports. It is important that any test is consistent and fair to all athletes to whom the test is administered. For this reason, WADA has its own code of ethics and ethics committee to oversee the activities of WADA and to ensure that any testing is transparent and accountable to the public.