Jaume Bosch

Growth hormone in sport: beyond Beijing 2008.

Human growth hormone (hGH) is a protein endogenously produced predominantly by the anterior pituitary gland. Native hGH and, especially, its recombinant analogue (rhGH), used to treat patients with hormone deficiency, are supposed to be abused by athletes searching its anabolic and lipolytic effects. Hence, hGH use has been prohibited for a long time by the sport authorities, but until recently, hGH abuse could not be detected. Two approaches have been followed when trying to develop methods for GH abuse detection. The direct method identifies an abnormal ratio between GH isoforms-a result of hGH exogenous administration. The time window to find a cheating athlete by this approach is limited by the excretion time of the hormone. The indirect approach measures serum biomarkers directly affected by GH intake (eg, markers of released liver growth factors and of bone and collagen turnover). In this approach, the retrospective power extends further. Alternative possibilities for cheating related to hGH could be the administration of recombinant growth factors themselves, the administration of hGH metabolic precursors such as ghrelin-like GH secretagogues, or the genetic manipulation of muscle growth-related genes (gene doping). In parallel with the new types of abuse, which will surely emerge in the near future, the research and development for the improvement of the analytical detection of GH itself will continue.

Characterisation of the 5 kDa growth hormone isoform

Objectives: The 5 kDa N-terminal fragment of 43 amino acids of human growth hormone (GH) shows a specific and significant in-vivo insulin-like activity. This isoform can be easily obtained by solid phase synthesis methods. Our objective in this study is to describe this procedure in detail and to provide structural information of the protein. Methods: Solid phase synthesis was employed for the synthesis of the 5 kDa GH isoform. Circular dichroism and limited proteolysis have been carried out to provide structural information about the folded state of the protein in solution. Surface plasmon resonance was used to compare the structural equivalence between the synthetic protein and a proteolytic homologue at an antibody binding level. For this purpose, a murine monoclonal antibody specific for the 5 kDa isoform was generated and characterised employing this and several other GH isoforms. Results: Circular dichroism and proteolysis results suggested that the C-terminal segment of the 5 kDa protein folds in an α-helix. The comparison of the synthetic product to its proteolytic homologue at an antibody binding level suggested structural equivalency. A highly specific antibody against the 5 kDa GH isoform was generated with null cross-reactivity for 17, 20 and 22 kDa isoforms. Kinetic data on the interaction with the synthetic 5 kDa GH was obtained. Conclusions: The structure of the protein appears to be different in comparison to when it is included within the 22 kDa GH isoform. Finally, a highly specific antibody has been generated. The possible significance of the 5 kDa protein as a potential agent for obesity-related diseases is discussed.

Analysis of urinary human growth hormone (hGH) using hydrogel nanoparticles and isoform differential immunoassays after short recombinant hGH treatment: preliminary results.

Successful application clinical-grade human growth hormone (hGH) immunoassays to the discovery of illegal doping cases has been rare. Indeed, the preferred biological matrix in doping control is urine, where the estimated baseline concentration of hGH falls well below the linear range and sensitivity threshold of all commercially available immunoassays, including hGH isoform differential immunoassays which can discriminate pituitary endogenous hGH from recombinant hGH. We employed hydrogel nanoparticles as a pre-processing step that concentrate urinary hGH into the linear range of isoform differential immunoassays. We explored the characteristics of immunoassays in urine spiked with both phGH or rhGH, after pre-treatment with the nanoparticles. Subsequently, pre-treatment was applied to urine obtained from 3 healthy volunteers administered during three days with daily subcutaneous injections of 0.026 mg/kg/day rhGH, Genotonorm(®). Linearity between both rhGH and phGH concentrations in urine measured by a chemoluminescent assay (Immulite) and in the particle eluate was evident for differential immunoassays (R square higher than 0.999). In case of treated individuals the recombinant/pituitary concentration ratios remained above the established World Anti-Doping Agency (WADA) criterion for hGH misuse up to 24h after the last administration dose, using both assays for volunteer 1 and 2 while in case of volunteer 3 results were inconclusive. The use of nanoparticles appears to open the possibility of assessing rhGH misuse in urine.

Tracking growth hormone abuse in sport: A comparison of distinct isoform-based assays

Detecting recombinant human growth hormone (rhGH) abuse in sport remains one of the major challenges in doping control. We have compared two different approaches to detect the hGH (human growth hormone) abuse. The first measures the concentrations of the 22 kDa hGH isoform (rec assay) and pituitary derived isoforms (pit assay) and a ratio rec/pit is obtained. The second measures the concentrations of 22 and 20 kDa hGH isoforms and also a ratio 22/20 kDa is derived. Using a single set (nine healthy male subjects, 7 days, 0.026 mg/kg/day of rhGH, 2 week wash out period) both approaches were compared. To quantify the agreement between the immunoassays, B.A. (Bland-Altman) analysis and P.r. (Pearson correlation) were used. To fully understand the assay readings, all relevant antibodies were characterised by surface plasmon resonance (SPR). In either approach the ratio numerator produces similar results and the denominator determines both signal-amplitude and time-frame of possible application. The rec vs pit approach displays a higher distinctive capacity to detect hGH abuse but the complex binding properties of the capture antibodies make it very difficult to evaluate the precise contributions of the individual hGH variants to the assay result. In the 22 vs 20 approach, the 20 kDa hGH concentration measures determine its applicability. Both approaches are based on a different principle, should be preferably applied within 24 h after rhGH administration, and are perfectly comparable given the results obtained. The reduced time frame of application indicates that their principle application should be preferably in an out-of-competition setting.

Surface plasmon resonance in doping analysis

AbstractDoping analysis relies on the determination of prohibited substances that should not be present in the body of an athlete or that should be below a threshold value. In the case of xenobiotics their mere presence is sufficient to establish a doping offence. However, in the case of human biotics the analytical method faces the difficulty of distinguishing between endogenous and exogenous origin. For this purpose ingenious strategies have been implemented, often aided by state-of-the-art technological advancements such as mass spectrometry in all its possible forms. For larger molecules, i.e. protein hormones, the innate structural complexity, the heterogeneous nature, and the extremely low levels in biological fluids have rendered the analytical procedures heavily dependent of immunological approaches. Although approaches these confer specificity and sensitivity to the applications, most rely on the use of two, or even three, antibody incubations with the consequent increment in assay variability. Moreover, the requirement for different antibodies that separately recognise different epitopes in screening and confirmation assays further contributes to differences encountered in either measurement. The development of analytical techniques to measure interactions directly, such as atomic force microscopy, quartz crystal microbalance or surface plasmon resonance, have greatly contributed to the accurate evaluation of molecular interactions in all fields of biology, and expectations are that this will only increase. Here, an overview is provided of surface plasmon resonance, and its particular value in application to the field of doping analysis. FigureSPR analysis to assist antidoping procedures

Tracking growth hormone abuse in sport: Performance of marker proteins in a controlled setting

Human growth hormone (hGH) abuse in sport is a challenge at present. The current strategy used, known as direct method, is based on the quantification of hGH variants in serum. An alternative strategy, known as indirect method, focuses on serum markers such as insulin-like growth factor I (IGF-I) and procollagen type III N-terminal propeptide (P-III-NP). The indirect method allows a longer window of detection (WOO) of hGH abuse. To evaluate the performance of the indirect method, in parallel to the direct method, a clinical trial with recombinant hGH (rhGH) was conducted on healthy male subjects during 7 days (0.026 mg(-1) kg(-1) person(-1) day(-1)). The data were fit to the discriminant formula proposed in the previously published GH-2000 project. The low sensitivity of the scores, judged from the high number of false negative outcomes, imposed a new discriminant analysis, standarised using local population subjects demographically similar to the ones of the study. The sensitivity of the method significantly increased, highlighting the importance of the standardisation. The indirect method allowed extended window of opportunity (WOO), although two false positive evaluations were observed derived from elevated basal IGF-I and P-III-NP concentrations stressing the need for an independent confirmation method. When direct and indirect methods were combined the best selectivity and sensitivity were achieved.

Determination of Recent Growth Hormone Abuse Using a Single Dried Blood Spot

BACKGROUND Although it is being increasingly applied, blood collection for drug testing in sport presents some logistic issues that complicate full applicability on a large scale. The use of dried blood spots (DBS) could benefit compliant blood testing considerably owing to its simplicity, minimal invasiveness, analyte stability, and reduced costs. The aim of this study was to evaluate the applicability of DBS to the methodology approved by the World Anti-Doping Agency (WADA) for detection of doping by recombinant human growth hormone (rhGH) in serum. METHODS A protocol for a single DBS analysis using the hGH isoforms differential immunoassays (kit 1 and kit 2) was developed and validated. A clinical study with healthy volunteers injected for 3 consecutive days with a low subcutaneous dose (0.027 mg · kg(-1) · day(-1) · person(-1)) of rhGH was conducted. Finger prick DBS and paired-time serum samples from arm venipuncture were compared. RESULTS The analysis of the DBS-based protocol indicated that with only a single blood spot it was possible to detect positivity for growth hormone abuse. In spite of the low rhGH dose administered and independently of the kit used, the window of detection for DBS was confirmed in all analyzed samples up to 8 h after rhGH administration and extended up to 12 h in 50% of the cases. Serum positivity was detected in all studied samples for 12 h after administration. CONCLUSIONS These results support the usefulness of DBS as a biological matrix for testing recent growth hormone abuse.

Growth hormone abuse and biological passport: is mannan-binding lectin a complementary candidate?

Objective:In the detection of human growth hormone (GH) abuse, the approach based on altered GH-related biomarkers is also being considered with respect to its application within the context of a biological passport. As a potential biomarker, mannan-binding lectin (MBL), which is reported to respond to recombinant GH (rGH) administration, is evaluated here. Design:Randomized and single blind and approved by the Ethical Committee (Comité Ético de Investigación Clínica-Instituto Municipal de Asistencia Sanitaria). Participants:One group of 12 male subjects (24.2 ± 2.2 years; 76.1 ± 6.1 kg) was studied. Interventions:Mannan-binding lectin concentration was measured in 12 healthy individuals after subcutaneous daily doses of 6 IU of rGH administration. Mannan-binding lectin serum concentration increased after rGH administration. Mannan-binding lectin concentration increases were observed 48 hours after the first administration and remained elevated for several days after the final dose. Main Outcome Measures:Mannan-binding lectin concentration increase and elapsed time to recover initial MBL values after the last rGH administration. Results:Absolute values displayed high interindividual variability, and 1 individual did not show any MBL increase (potential MBL deficiency). Mannan-binding lectin protein showed a clear concentration increase after continued rGH administration, despite the high heterogeneity found between individuals. Conclusions:The use of MBL as a complementary GH-related biomarker could be of interest, taking advantage of the high increases (up to 700%) and the relatively slow recovery time.