Drug Testing and Analysis
Short communication Received: 9 March 2014
Revised: 11 April 2014
Accepted: 11 April 2014
Published online in Wiley Online Library: 14 May 2014
(www.drugtestinganalysis.com) DOI 10.1002/dta.1666
The effect of a period of intense exercise on the marker approach to detect growth hormone doping in sports Sven Christian Voss,a*† Neil Robinson,b Mohammed Alsayrafi,a Pitre C. Bourdon,c Yorck Olaf Schumacher,d Martial Saugyb and Sylvain Giraudb† The major objective of this study was to investigate the effects of several days of intense exercise on the growth hormone marker approach to detect doping with human growth hormone (hGH). In addition we investigated the effect of changes in plasma volume on the test. Fifteen male athletes performed a simulated nine-day cycling stage race. Blood samples were collected twice daily over a period of 15 days (stage race + three days before and after). Plasma volumes were estimated by the optimized CO Rebreathing method. IGF-1 and P-III-NP were analyzed by Siemens Immulite and Cisbio Assays, respectively. All measured GH 2000 scores were far below the published decision limits for an adverse analytical finding. The period of exercise did not increase the GH-scores; however the accompanying effect of the increase in Plasma Volume yielded in essentially lower GH-scores. We could demonstrate that a period of heavy, long-term exercise with changes in plasma volume does not interfere with the decision limits for an adverse analytical finding. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: doping; hGH; IGF-1; passport; plasma volume; GH-score; exercise
Introduction
582
Human growth hormone (hGH) is a peptide hormone with anabolic and performance enhancing effects.[1–3] There is evidence that it is misused by recreational athletes[4] and positive doping cases also prove its use in elite sport. It is secreted by the pituitary gland, acts on the liver to produce insulin-like growth factor 1 (IGF-1) and also stimulates soft tissue and bones to release type 3 Procollagen (P-III-nP). While hGH preparations were previously extracted from the pituitary gland of human cadavers, it is nowadays mainly produced through genetic engineering.[5] The World Anti-Doping Agency (WADA) currently has hGH on its prohibited list under S2 – Peptide Hormones, Growth Factors and Related Substances; however its use in sport has been forbidden by the International Olympic Committee (IOC) since 1989. At the 2012 Olympic Games in London, the marker approach to detect hGH abuse was added to the repertoire of anti-doping tests, which was so far limited to the isoform method.[6] The marker approach is the result of the GH 2000 project, a large multi-centre study which was supported by the IOC, the European Union, and two hGH manufacturers.[7] Testing is based on the concentration of the two markers IGF-1 and P-III-nP, which both increase following hGH administration. Decision limits for the current commercial IGF-1 and P-III-nP assays were published shortly before the Olympics after performing a study including 404 male and 94 female elite athletes.[8] Currently these decision limits are only available for results obtained by four assays. These assays are the Immulite IGF-I assay (Siemens, Llanberis, UK), the A15729 IGF-I IRMA (Immunotech SAS, Marseille, France), the RIA-gnost P-III-P (Cisbio, Gif-sur-Yvette, France) and the UniQ™ P-III-NP RIA (Orion Diagnostica, Espoo, Finland). The scores are calculated using the following published equations where log is the natural logarithm.
Drug Test. Analysis 2014, 6, 582–586
GH-2000 Score ðMaleÞ : 6:586 þ 2:905 logðP-III-PÞ þ2:100 logðIGF-1Þ 101:737=age
(1)
GH-2000 Score ðFemaleÞ : 8:459 þ 2:454 logðP-III-PÞ þ2:195 log ðIGF-1Þ 73:666=age
(2)
The decision limit for male athletes and the assays applied in this study lies for a combined test specificity of 1 in 10 000 at 2.69. The effect of acute exercise (duration of up to 30 min) on the hGH markers has previously been evaluated by Ehrnborg[9] and Wallace,[10,11] however the IGF-1 assays used in their studies were different from that used in current anti-doping analysis. In addition all studies to date have only investigated the effect of acute exercise on the GH 2000 markers while the effects of longer periods of intensive physical exercise remain unexplored, despite the fact that many sporting events such as cycling stage races are often multi-day events where the athlete has to produce intensive effort over several days.
* Correspondence to: Sven Christian Voss, Anti-Doping Lab Qatar, PO Box 27775, Doha, Aspire Zone, Doha, Qatar. E-mail: [email protected] †
These authors contributed equally to this work.
a Anti-Doping Lab Qatar, PO Box 27775, Doha, Aspire Zone, Doha, Qatar b Swiss Laboratory for Doping Analyses, University Centre of Legal Medicine, Geneva and Lausanne, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland c Aspire Academy for Sports Excellence-Sport Science Department, Doha, Qatar d Aspetar Orthopedic and Sports Medicine Hospital, Doha, Qatar
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Testing and Analysis
Effect of exercise on anti-doping GH marker test Thus, the major goal of our study was to investigate the effect of several days of intensive exercise on the hGH marker approach with test kits currently used in anti-doping taking in consideration the actual WADA guidelines for blood sample collection. Additionally the effect of changes in plasma volume was investigated.
Methodology Subjects Fifteen healthy male Caucasian amateur cyclists and triathletes took part in this study. The athletes were recruited through an advertisement at the university. To assure subjects were really drug free, they were also tested on daily base by the WADA hGH isoform test. For further information regarding anthropometric and fitness data please refer to Voss et al.[12] The study was conducted according to the Helsinki protocol and approved by the ethics committee of the University of Freiburg in
Germany. Written informed consent was obtained from all athletes before the start of the study. Cycling intervention To mimic a real life training and competition regime, the subjects performed a three-day resting period (travel and tapering phase) followed by a nine-day cycling stage race simulation and a threeday recovery period. The simulated stage race was designed to be as close to a real race as possible, with certain tasks performed by the participants every day as already previously described.[12] Special financial primes for different tasks were offered each day to increase motivation. Blood sampling Venous blood sampling was performed according to the current WADA guidelines for the athlete biological passport.[13] Samples
Drug Test. Analysis 2014, 6, 582–586
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
583
Figure 1. Circles give the GH score for each collection point. The dotted line shows the Decision limit described by Erotokritou-Mulligan et al.[8]
Drug Testing and Analysis
S. C. Voss et al.
were collected every morning before breakfast at 8.00 am and every evening at 6.00 pm (which was at least two hours after exercise) over the 15-day study period. A total of 11.7 mL blood was drawn at each collection (one 2.7 mL EDTA tube (Sarstedt, Numbrecht, Germany) and one 9.0 mL tube with clot activator (S-Monovette, Sarstedt, Numbrecht, Germany)).
the values within 25–75% of the population and the line in the box is the median. Outliers are presented as open circles (1.5x interquartile range) or stars (3x interquartile range).
Haematological variables and plasma volume (PV)
The combined decision limit for the applied test kits have been set at 2.69 which represents the individual specificity of 1 in 10 000.[8] All measured GH 2000 scores in our study were far below this limit. The highest values reached by two subjects were GH-scores of 1.30 and 1.31. Subject 12 presented the biggest variation with GH-scores from 1.11 to 1.30 while the whole study population showed a variation range from 2.81 to 1.31. Figure 1 presents the individual range of GH-scores for each subject during the 15 study days. As demonstrated in Figure 2, we observed significant higher GH-scores (p < 0.0001) in the morning when compared to the evening values. The mean absolute GH-score difference was 0.34, Subject 12 had the highest intra-day GH-score difference with 1.32. The Box-Plots in Figure 3 present the daily GH-scores. After the initial resting phase (days 1–3), the race period starts (days 4–12). Days 6–8 of this period show the lowest GH-scores, which correlates to the observed plasma volumes which showed the highest values on these days.[15] GH-scores at the lowest and highest plasma volume levels were significantly different (p = 0.001) as shown in Figure 4. The actual decision limits of the subjects in this study were however not affected. There might be a risk for athletes with naturally very high values to pass the cut off limit for a negative sample when dehydrated. This could however be eliminated by introducing an individual threshold as already applied in the haematological and steroidal modules of the biological passport.
The EDTA blood samples were analyzed within 90 min of withdrawal on a Sysmex XT-2000i haematology analyzer (Sysmex Corp., Kobe, Japan) according to WADA guidelines. To investigate PV effects, haemoglobin mass was measured using the optimized CO rebreathing method as previously described by Schmidt and Prommer.[14] Briefly the method consists of a 2 min rebreathing manoeuvre with an administration of 1 mL CO per kg bodyweight. Before and at defined time points after the CO administration, the CO-haemoglobin concentration was measured by a blood gas analyzer. Haemoglobin mass was measured in each subject in duplicate at the beginning of the study. The true haemoglobin mass for each time point was calculated taking into account the exact loss of haemoglobin due to blood sampling.[15] PV for each sampling time point was then calculated by the following formulas: Blood Volume ðmLÞ ¼ Haemoglobin mass ðgÞ=Haemoglobin ðg=dLÞ • 100
(3) Red Cell Volume ðmLÞ ¼ Blood Volume ðmlÞ • Haematocrit ð%Þ
(4)
Plasma Volume ðmLÞ ¼ Blood Volume–Red Cell Volume
(5)
Serum variables Blood was collected following the international standard for testing using Sarstedt S Monovette tubes.[16] Samples were centrifuged on-site after clotting and aliquots were then stored at 20°C. IGF-1was tested on an Immulite 2000 (Siemens Healthcare Diagnostics SA, Zürich, Switzerland), while P-III-nP was tested by a manual RIA test (Procollagen III peptide RIAs assay; CisBio Bioassays. Codolet, France ). No morning serum samples were withdrawn on Day 1 (travel day) so in total 29 serum samples were collected for each subject. However due to occasional low sample volumes it was not possible to analyze all 29 samples per subject (Figure 1).
Results
Discussion Our data support the GH score decision limit of 2.69. None of our subjects came close to this limit. We calculated that with our
Statistical analyses
584
All statistics were performed using Excel 2010 (Microsoft) and SPSS 20 (IBM) software. Datasets were controlled for normal distribution by the Kolmogorov-Smirnoff Test. To determine whether plasma volume could have an effect on the marker score, the data for each subject at the lowest and at the highest plasma volume were analyzed for significance by the Wilcoxon signed-rank test, which was conducted at a 95% confidence interval. To compare results from samples taken in the morning with samples taken in the evening only data of the subjects where values for both time points were available (n = 195) were used in the statistics. As the values were normally distributed a paired t-test was applied. Each box plot uses the same definition. Whiskers present the 95% confidence interval, the Box defines
wileyonlinelibrary.com/journal/dta
Figure 2. GH Scores in the Morning and Evening were significantly different (p < 0.0001). There was no relation between duration of exercise and the GH-Score.
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 582–586
Drug Testing and Analysis
Effect of exercise on anti-doping GH marker test
Figure 3. GH-Score values presented as box-plots over the complete 15 day period of the study.
Figure 4. GH-Scores measured at the highest (4189 ± 456 mL) and at the lowest (3208 ± 369 mL) Plasma Volume of each Subject were significantly different (p = 0.001). The mean difference was 0.80 units while the median change is only 0.43. The boxplot for the considered GH-score population is also shown.
Drug Test. Analysis 2014, 6, 582–586
Conclusion Research on existing procedures in sports drug testing is an important role of doping control laboratories in order to enable advanced analyses and ensure confidence in analytical results. The results of the present study showed that the effects of heavy, long-term exercise do not appear to interfere with the decision limits for an adverse analytical finding.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
585
highest mean score (0.18) and the highest standard deviation (0.59), the specificity of this decision limit is better than 1/10 000. Comparing all GH-score variance (0.46) to the mean variance of all subjects (0.16) indicates that the inter-individual variance is bigger than the intra-individual one. Even the maximal observed variance for one subject is only 0.33 which is in favour of an individual decision limit. Our results indicate that there is a weak correlation between PV and GH-score; for a precise estimation, however, more data would be needed. It should be a future aim to obtain more information about the uncertainty that PV can add on the GH-score and include this factor in the passport to set an individual decision limit.
A 9-day exercise period with several hours of exercise per day, did not increase the subjects’ individual GH-scores. On the contrary we could observe a trend to decreased GH-scores during the exercise period, as visible in Figure 3, days 6–8. This phenomenon can be most probably explained by the increased plasma volume which occurs as a result of the adaptation to the exercise.[17,18] Our data regarding the diurnal variation showed a highly significant difference between morning and evening values which is in contradiction to the expectations which derive from Holts paper who describes a low intra-day variation for IGF-1 and P-III-nP.[19] Possible explanations for this could be the by the extreme exercise induced plasma volume in this study as well as overnight dehydration and postural issues resulting in a slightly decreased plasma volume and thus haemoconcentration in the morning, which resolves progressively over the day. Compared to our study, the above mentioned previous single exercise related studies do not report any GH-scores but only the concentrations of IGF-1 and P-III-nP.[9–11] These studies show that no increases of IGF-1 and P-III-nP could be observed when the sample is taken 2 h after an exercise protocol. Although the assays applied in these studies are different from the recommended assays today and the exercise is shorter than in our study the findings also support the stability of the marker approach.
Drug Testing and Analysis
S. C. Voss et al.
Acknowledgements This project was funded by Anti-Doping Lab Qatar, Anti-Doping Switzerland, Aspire Academy and UK Anti-Doping. The authors would also like to gratefully acknowledge; Anti-Doping laboratories of Qatar and Switzerland for support, and Magali Wicht, Michaël Goy, Noora Alsowaidi, Mohamed Ibrahim Elzain Elgingo, Felix Klodt and Damiano Nonis for their help in planning and technical assistance. We also acknowledge Siemens Healthcare Diagnostics SA (Switzerland) for providing the IGF-1 kits.
References [1] M.L. Healy, J. Gibney, D.L. Russell-Jones, C. Pentecost, P. Croos, P.H. Sonksen, A.M. Umpleby. High dose growth hormone exerts an anabolic effect at rest and during exercise in endurance-trained athletes. J. Clin. Endocr. Metab. 2003, 88, 5221. [2] M.R. Graham, J.S. Baker, P. Evans, A. Kicman, D. Cowan, D. Hullin, N. Thomas, B. Davies. Physical effects of short-term recombinant human growth hormone administration in abstinent steroid dependency. Horm. Res. 2008, 69, 343. [3] U. Meinhardt, A.E. Nelson, J.L. Hansen, V. Birzniece, D. Clifford, K.C. Leung, K. Graham, K.K. Ho. The effects of growth hormone on body composition and physical performance in recreational athletes: A randomized trial. Ann. Intern. Med. 2010, 152, 568. [4] B.P. Brennan, G. Kanayama, J.I. Hudson, H.G. Pope. Human growth hormone abuse in male weightlifters. Am. J. Addict. 2011, 20, 9. [5] M. Saugy, N. Robinson, C. Saudan, N. Baume, L. Avois, P. Mangin. Human growth hormone doping in sport. Brit. J. Sports Med. 2006, 40, 35. [6] M. Bidlingmaier, J. Manolopoulo. Detecting growth hormone abuse in athletes. Endocr. Metab. Clin. 2010, 39, 25. [7] R.I. Holt, I. Erotokritou-Mulligan, P.H. Sonksen. The history of doping and growth hormone abuse in sport. Growth Horm. IGF Res. 2009, 19, 320. [8] I. Erotokritou-Mulligan, N. Guha, M. Stow, E.E. Bassett, C. Bartlett, D.A. Cowan, P.H. Sonksen, R.I. Holt. The development of decision limits for the implementation of the GH-2000 detection methodology using current commercial insulin-like growth factor-I and aminoterminal pro-peptide of type III collagen assays. Growth Horm. IGF Res. 2012, 22, 53.
[9] C. Ehrnborg, K.H. Lange, R. Dall, J.S. Christiansen, P.A. Lundberg, R.C. Baxter, M.A. Boroujerdi, B.A. Bengtsson, M.L. Healey, C. Pentecost, S. Longobardi, R. Napoli, T. Rosen. The growth hormone/insulin-like growth factor-I axis hormones and bone markers in elite athletes in response to a maximum exercise test. J. Clin. Endocr. Metab. 2003, 88, 394. [10] J.D. Wallace, R.C. Cuneo, R. Baxter, H. Orskov, N. Keay, C. Pentecost, R. Dall, T. Rosen, J.O. Jorgensen, A. Cittadini, S. Longobardi, L. Sacca, J.S. Christiansen, B.A. Bengtsson, P.H. Sonksen. Responses of the growth hormone (GH) and insulin-like growth factor axis to exercise, GH administration, and GH withdrawal in trained adult males: a potential test for GH abuse in sport. J. Clin. Endocr. Metab. 1999, 84, 3591. [11] J.D. Wallace, R.C. Cuneo, P.A. Lundberg, T. Rosen, J.O. Jorgensen, S. Longobardi, N. Keay, L. Sacca, J.S. Christiansen, B.A. Bengtsson, P.H. Sonksen. Responses of markers of bone and collagen turnover to exercise, growth hormone (GH) administration, and GH withdrawal in trained adult males. J. Clin. Endocr. Metab. 2000, 85, 124. [12] S.C. Voss, S. Giraud, M. Alsayrafi, P.C. Bourdon, Y.O. Schumacher, M. Saugy, N. Robinson. The effect of a period of intensive exercise on the isoform test to detect growth hormone doping in sports. Growth Horm. IGF Res. 2013, 23, 105. [13] WADA. Athelete Biological Passort Operating Guidelines. Available at: http://www.wada-ama.org/Documents/Science_Medicine/Athlete_Bio logical_Passport/WADA-ABP-Operating-Guidelines_v3.1-EN.pdf [9 April 2012]. [14] W. Schmidt, N. Prommer. The optimised CO-rebreathing method: A new tool to determine total haemoglobin mass routinely. Eur. J. Appl. Physiol. 2005, 95, 486. [15] S.C. Voss, M. Alsayrafi, P.C. Bourdon, F. Klodt, D. Nonis, W.G. Hopkins, Y. O. Schumacher. Variability of serum markers of erythropoiesis during 6 days of racing in highly trained cyclists. Int. J. Sports Med. 2013, 35, 89. [16] WADA. The World Anti-Doping Code, International Standard for Testing. Available at: www.wada-ama.org/Documents/World_AntiDoping_Program/WADP-IS-Testing/2012/WADA_IST_2012_EN.pdf [9 April 2012] [17] V.A. Convertino. Blood volume response to physical activity and inactivity. Am. J. Med. Sci. 2007, 334, 72. [18] Y.O. Schumacher, F. Klodt, D. Nonis, T. Pottgiesser, M. Alsayrafi, P.C. Bourdon, S.C. Voss. The impact of long-haul air travel on variables of the athlete’s biological passport. Int. J. Lab. Hematol. 2012, 34, 641. [19] R.I. Holt. Detecting growth hormone abuse in athletes. Drug Test. Anal. 2009, 1, 426.
586 wileyonlinelibrary.com/journal/dta
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 582–586
Short communication Received: 9 March 2014
Revised: 11 April 2014
Accepted: 11 April 2014
Published online in Wiley Online Library: 14 May 2014
(www.drugtestinganalysis.com) DOI 10.1002/dta.1666
The effect of a period of intense exercise on the marker approach to detect growth hormone doping in sports Sven Christian Voss,a*† Neil Robinson,b Mohammed Alsayrafi,a Pitre C. Bourdon,c Yorck Olaf Schumacher,d Martial Saugyb and Sylvain Giraudb† The major objective of this study was to investigate the effects of several days of intense exercise on the growth hormone marker approach to detect doping with human growth hormone (hGH). In addition we investigated the effect of changes in plasma volume on the test. Fifteen male athletes performed a simulated nine-day cycling stage race. Blood samples were collected twice daily over a period of 15 days (stage race + three days before and after). Plasma volumes were estimated by the optimized CO Rebreathing method. IGF-1 and P-III-NP were analyzed by Siemens Immulite and Cisbio Assays, respectively. All measured GH 2000 scores were far below the published decision limits for an adverse analytical finding. The period of exercise did not increase the GH-scores; however the accompanying effect of the increase in Plasma Volume yielded in essentially lower GH-scores. We could demonstrate that a period of heavy, long-term exercise with changes in plasma volume does not interfere with the decision limits for an adverse analytical finding. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: doping; hGH; IGF-1; passport; plasma volume; GH-score; exercise
Introduction
582
Human growth hormone (hGH) is a peptide hormone with anabolic and performance enhancing effects.[1–3] There is evidence that it is misused by recreational athletes[4] and positive doping cases also prove its use in elite sport. It is secreted by the pituitary gland, acts on the liver to produce insulin-like growth factor 1 (IGF-1) and also stimulates soft tissue and bones to release type 3 Procollagen (P-III-nP). While hGH preparations were previously extracted from the pituitary gland of human cadavers, it is nowadays mainly produced through genetic engineering.[5] The World Anti-Doping Agency (WADA) currently has hGH on its prohibited list under S2 – Peptide Hormones, Growth Factors and Related Substances; however its use in sport has been forbidden by the International Olympic Committee (IOC) since 1989. At the 2012 Olympic Games in London, the marker approach to detect hGH abuse was added to the repertoire of anti-doping tests, which was so far limited to the isoform method.[6] The marker approach is the result of the GH 2000 project, a large multi-centre study which was supported by the IOC, the European Union, and two hGH manufacturers.[7] Testing is based on the concentration of the two markers IGF-1 and P-III-nP, which both increase following hGH administration. Decision limits for the current commercial IGF-1 and P-III-nP assays were published shortly before the Olympics after performing a study including 404 male and 94 female elite athletes.[8] Currently these decision limits are only available for results obtained by four assays. These assays are the Immulite IGF-I assay (Siemens, Llanberis, UK), the A15729 IGF-I IRMA (Immunotech SAS, Marseille, France), the RIA-gnost P-III-P (Cisbio, Gif-sur-Yvette, France) and the UniQ™ P-III-NP RIA (Orion Diagnostica, Espoo, Finland). The scores are calculated using the following published equations where log is the natural logarithm.
Drug Test. Analysis 2014, 6, 582–586
GH-2000 Score ðMaleÞ : 6:586 þ 2:905 logðP-III-PÞ þ2:100 logðIGF-1Þ 101:737=age
(1)
GH-2000 Score ðFemaleÞ : 8:459 þ 2:454 logðP-III-PÞ þ2:195 log ðIGF-1Þ 73:666=age
(2)
The decision limit for male athletes and the assays applied in this study lies for a combined test specificity of 1 in 10 000 at 2.69. The effect of acute exercise (duration of up to 30 min) on the hGH markers has previously been evaluated by Ehrnborg[9] and Wallace,[10,11] however the IGF-1 assays used in their studies were different from that used in current anti-doping analysis. In addition all studies to date have only investigated the effect of acute exercise on the GH 2000 markers while the effects of longer periods of intensive physical exercise remain unexplored, despite the fact that many sporting events such as cycling stage races are often multi-day events where the athlete has to produce intensive effort over several days.
* Correspondence to: Sven Christian Voss, Anti-Doping Lab Qatar, PO Box 27775, Doha, Aspire Zone, Doha, Qatar. E-mail: [email protected] †
These authors contributed equally to this work.
a Anti-Doping Lab Qatar, PO Box 27775, Doha, Aspire Zone, Doha, Qatar b Swiss Laboratory for Doping Analyses, University Centre of Legal Medicine, Geneva and Lausanne, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland c Aspire Academy for Sports Excellence-Sport Science Department, Doha, Qatar d Aspetar Orthopedic and Sports Medicine Hospital, Doha, Qatar
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Testing and Analysis
Effect of exercise on anti-doping GH marker test Thus, the major goal of our study was to investigate the effect of several days of intensive exercise on the hGH marker approach with test kits currently used in anti-doping taking in consideration the actual WADA guidelines for blood sample collection. Additionally the effect of changes in plasma volume was investigated.
Methodology Subjects Fifteen healthy male Caucasian amateur cyclists and triathletes took part in this study. The athletes were recruited through an advertisement at the university. To assure subjects were really drug free, they were also tested on daily base by the WADA hGH isoform test. For further information regarding anthropometric and fitness data please refer to Voss et al.[12] The study was conducted according to the Helsinki protocol and approved by the ethics committee of the University of Freiburg in
Germany. Written informed consent was obtained from all athletes before the start of the study. Cycling intervention To mimic a real life training and competition regime, the subjects performed a three-day resting period (travel and tapering phase) followed by a nine-day cycling stage race simulation and a threeday recovery period. The simulated stage race was designed to be as close to a real race as possible, with certain tasks performed by the participants every day as already previously described.[12] Special financial primes for different tasks were offered each day to increase motivation. Blood sampling Venous blood sampling was performed according to the current WADA guidelines for the athlete biological passport.[13] Samples
Drug Test. Analysis 2014, 6, 582–586
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
583
Figure 1. Circles give the GH score for each collection point. The dotted line shows the Decision limit described by Erotokritou-Mulligan et al.[8]
Drug Testing and Analysis
S. C. Voss et al.
were collected every morning before breakfast at 8.00 am and every evening at 6.00 pm (which was at least two hours after exercise) over the 15-day study period. A total of 11.7 mL blood was drawn at each collection (one 2.7 mL EDTA tube (Sarstedt, Numbrecht, Germany) and one 9.0 mL tube with clot activator (S-Monovette, Sarstedt, Numbrecht, Germany)).
the values within 25–75% of the population and the line in the box is the median. Outliers are presented as open circles (1.5x interquartile range) or stars (3x interquartile range).
Haematological variables and plasma volume (PV)
The combined decision limit for the applied test kits have been set at 2.69 which represents the individual specificity of 1 in 10 000.[8] All measured GH 2000 scores in our study were far below this limit. The highest values reached by two subjects were GH-scores of 1.30 and 1.31. Subject 12 presented the biggest variation with GH-scores from 1.11 to 1.30 while the whole study population showed a variation range from 2.81 to 1.31. Figure 1 presents the individual range of GH-scores for each subject during the 15 study days. As demonstrated in Figure 2, we observed significant higher GH-scores (p < 0.0001) in the morning when compared to the evening values. The mean absolute GH-score difference was 0.34, Subject 12 had the highest intra-day GH-score difference with 1.32. The Box-Plots in Figure 3 present the daily GH-scores. After the initial resting phase (days 1–3), the race period starts (days 4–12). Days 6–8 of this period show the lowest GH-scores, which correlates to the observed plasma volumes which showed the highest values on these days.[15] GH-scores at the lowest and highest plasma volume levels were significantly different (p = 0.001) as shown in Figure 4. The actual decision limits of the subjects in this study were however not affected. There might be a risk for athletes with naturally very high values to pass the cut off limit for a negative sample when dehydrated. This could however be eliminated by introducing an individual threshold as already applied in the haematological and steroidal modules of the biological passport.
The EDTA blood samples were analyzed within 90 min of withdrawal on a Sysmex XT-2000i haematology analyzer (Sysmex Corp., Kobe, Japan) according to WADA guidelines. To investigate PV effects, haemoglobin mass was measured using the optimized CO rebreathing method as previously described by Schmidt and Prommer.[14] Briefly the method consists of a 2 min rebreathing manoeuvre with an administration of 1 mL CO per kg bodyweight. Before and at defined time points after the CO administration, the CO-haemoglobin concentration was measured by a blood gas analyzer. Haemoglobin mass was measured in each subject in duplicate at the beginning of the study. The true haemoglobin mass for each time point was calculated taking into account the exact loss of haemoglobin due to blood sampling.[15] PV for each sampling time point was then calculated by the following formulas: Blood Volume ðmLÞ ¼ Haemoglobin mass ðgÞ=Haemoglobin ðg=dLÞ • 100
(3) Red Cell Volume ðmLÞ ¼ Blood Volume ðmlÞ • Haematocrit ð%Þ
(4)
Plasma Volume ðmLÞ ¼ Blood Volume–Red Cell Volume
(5)
Serum variables Blood was collected following the international standard for testing using Sarstedt S Monovette tubes.[16] Samples were centrifuged on-site after clotting and aliquots were then stored at 20°C. IGF-1was tested on an Immulite 2000 (Siemens Healthcare Diagnostics SA, Zürich, Switzerland), while P-III-nP was tested by a manual RIA test (Procollagen III peptide RIAs assay; CisBio Bioassays. Codolet, France ). No morning serum samples were withdrawn on Day 1 (travel day) so in total 29 serum samples were collected for each subject. However due to occasional low sample volumes it was not possible to analyze all 29 samples per subject (Figure 1).
Results
Discussion Our data support the GH score decision limit of 2.69. None of our subjects came close to this limit. We calculated that with our
Statistical analyses
584
All statistics were performed using Excel 2010 (Microsoft) and SPSS 20 (IBM) software. Datasets were controlled for normal distribution by the Kolmogorov-Smirnoff Test. To determine whether plasma volume could have an effect on the marker score, the data for each subject at the lowest and at the highest plasma volume were analyzed for significance by the Wilcoxon signed-rank test, which was conducted at a 95% confidence interval. To compare results from samples taken in the morning with samples taken in the evening only data of the subjects where values for both time points were available (n = 195) were used in the statistics. As the values were normally distributed a paired t-test was applied. Each box plot uses the same definition. Whiskers present the 95% confidence interval, the Box defines
wileyonlinelibrary.com/journal/dta
Figure 2. GH Scores in the Morning and Evening were significantly different (p < 0.0001). There was no relation between duration of exercise and the GH-Score.
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 582–586
Drug Testing and Analysis
Effect of exercise on anti-doping GH marker test
Figure 3. GH-Score values presented as box-plots over the complete 15 day period of the study.
Figure 4. GH-Scores measured at the highest (4189 ± 456 mL) and at the lowest (3208 ± 369 mL) Plasma Volume of each Subject were significantly different (p = 0.001). The mean difference was 0.80 units while the median change is only 0.43. The boxplot for the considered GH-score population is also shown.
Drug Test. Analysis 2014, 6, 582–586
Conclusion Research on existing procedures in sports drug testing is an important role of doping control laboratories in order to enable advanced analyses and ensure confidence in analytical results. The results of the present study showed that the effects of heavy, long-term exercise do not appear to interfere with the decision limits for an adverse analytical finding.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
585
highest mean score (0.18) and the highest standard deviation (0.59), the specificity of this decision limit is better than 1/10 000. Comparing all GH-score variance (0.46) to the mean variance of all subjects (0.16) indicates that the inter-individual variance is bigger than the intra-individual one. Even the maximal observed variance for one subject is only 0.33 which is in favour of an individual decision limit. Our results indicate that there is a weak correlation between PV and GH-score; for a precise estimation, however, more data would be needed. It should be a future aim to obtain more information about the uncertainty that PV can add on the GH-score and include this factor in the passport to set an individual decision limit.
A 9-day exercise period with several hours of exercise per day, did not increase the subjects’ individual GH-scores. On the contrary we could observe a trend to decreased GH-scores during the exercise period, as visible in Figure 3, days 6–8. This phenomenon can be most probably explained by the increased plasma volume which occurs as a result of the adaptation to the exercise.[17,18] Our data regarding the diurnal variation showed a highly significant difference between morning and evening values which is in contradiction to the expectations which derive from Holts paper who describes a low intra-day variation for IGF-1 and P-III-nP.[19] Possible explanations for this could be the by the extreme exercise induced plasma volume in this study as well as overnight dehydration and postural issues resulting in a slightly decreased plasma volume and thus haemoconcentration in the morning, which resolves progressively over the day. Compared to our study, the above mentioned previous single exercise related studies do not report any GH-scores but only the concentrations of IGF-1 and P-III-nP.[9–11] These studies show that no increases of IGF-1 and P-III-nP could be observed when the sample is taken 2 h after an exercise protocol. Although the assays applied in these studies are different from the recommended assays today and the exercise is shorter than in our study the findings also support the stability of the marker approach.
Drug Testing and Analysis
S. C. Voss et al.
Acknowledgements This project was funded by Anti-Doping Lab Qatar, Anti-Doping Switzerland, Aspire Academy and UK Anti-Doping. The authors would also like to gratefully acknowledge; Anti-Doping laboratories of Qatar and Switzerland for support, and Magali Wicht, Michaël Goy, Noora Alsowaidi, Mohamed Ibrahim Elzain Elgingo, Felix Klodt and Damiano Nonis for their help in planning and technical assistance. We also acknowledge Siemens Healthcare Diagnostics SA (Switzerland) for providing the IGF-1 kits.
References [1] M.L. Healy, J. Gibney, D.L. Russell-Jones, C. Pentecost, P. Croos, P.H. Sonksen, A.M. Umpleby. High dose growth hormone exerts an anabolic effect at rest and during exercise in endurance-trained athletes. J. Clin. Endocr. Metab. 2003, 88, 5221. [2] M.R. Graham, J.S. Baker, P. Evans, A. Kicman, D. Cowan, D. Hullin, N. Thomas, B. Davies. Physical effects of short-term recombinant human growth hormone administration in abstinent steroid dependency. Horm. Res. 2008, 69, 343. [3] U. Meinhardt, A.E. Nelson, J.L. Hansen, V. Birzniece, D. Clifford, K.C. Leung, K. Graham, K.K. Ho. The effects of growth hormone on body composition and physical performance in recreational athletes: A randomized trial. Ann. Intern. Med. 2010, 152, 568. [4] B.P. Brennan, G. Kanayama, J.I. Hudson, H.G. Pope. Human growth hormone abuse in male weightlifters. Am. J. Addict. 2011, 20, 9. [5] M. Saugy, N. Robinson, C. Saudan, N. Baume, L. Avois, P. Mangin. Human growth hormone doping in sport. Brit. J. Sports Med. 2006, 40, 35. [6] M. Bidlingmaier, J. Manolopoulo. Detecting growth hormone abuse in athletes. Endocr. Metab. Clin. 2010, 39, 25. [7] R.I. Holt, I. Erotokritou-Mulligan, P.H. Sonksen. The history of doping and growth hormone abuse in sport. Growth Horm. IGF Res. 2009, 19, 320. [8] I. Erotokritou-Mulligan, N. Guha, M. Stow, E.E. Bassett, C. Bartlett, D.A. Cowan, P.H. Sonksen, R.I. Holt. The development of decision limits for the implementation of the GH-2000 detection methodology using current commercial insulin-like growth factor-I and aminoterminal pro-peptide of type III collagen assays. Growth Horm. IGF Res. 2012, 22, 53.
[9] C. Ehrnborg, K.H. Lange, R. Dall, J.S. Christiansen, P.A. Lundberg, R.C. Baxter, M.A. Boroujerdi, B.A. Bengtsson, M.L. Healey, C. Pentecost, S. Longobardi, R. Napoli, T. Rosen. The growth hormone/insulin-like growth factor-I axis hormones and bone markers in elite athletes in response to a maximum exercise test. J. Clin. Endocr. Metab. 2003, 88, 394. [10] J.D. Wallace, R.C. Cuneo, R. Baxter, H. Orskov, N. Keay, C. Pentecost, R. Dall, T. Rosen, J.O. Jorgensen, A. Cittadini, S. Longobardi, L. Sacca, J.S. Christiansen, B.A. Bengtsson, P.H. Sonksen. Responses of the growth hormone (GH) and insulin-like growth factor axis to exercise, GH administration, and GH withdrawal in trained adult males: a potential test for GH abuse in sport. J. Clin. Endocr. Metab. 1999, 84, 3591. [11] J.D. Wallace, R.C. Cuneo, P.A. Lundberg, T. Rosen, J.O. Jorgensen, S. Longobardi, N. Keay, L. Sacca, J.S. Christiansen, B.A. Bengtsson, P.H. Sonksen. Responses of markers of bone and collagen turnover to exercise, growth hormone (GH) administration, and GH withdrawal in trained adult males. J. Clin. Endocr. Metab. 2000, 85, 124. [12] S.C. Voss, S. Giraud, M. Alsayrafi, P.C. Bourdon, Y.O. Schumacher, M. Saugy, N. Robinson. The effect of a period of intensive exercise on the isoform test to detect growth hormone doping in sports. Growth Horm. IGF Res. 2013, 23, 105. [13] WADA. Athelete Biological Passort Operating Guidelines. Available at: http://www.wada-ama.org/Documents/Science_Medicine/Athlete_Bio logical_Passport/WADA-ABP-Operating-Guidelines_v3.1-EN.pdf [9 April 2012]. [14] W. Schmidt, N. Prommer. The optimised CO-rebreathing method: A new tool to determine total haemoglobin mass routinely. Eur. J. Appl. Physiol. 2005, 95, 486. [15] S.C. Voss, M. Alsayrafi, P.C. Bourdon, F. Klodt, D. Nonis, W.G. Hopkins, Y. O. Schumacher. Variability of serum markers of erythropoiesis during 6 days of racing in highly trained cyclists. Int. J. Sports Med. 2013, 35, 89. [16] WADA. The World Anti-Doping Code, International Standard for Testing. Available at: www.wada-ama.org/Documents/World_AntiDoping_Program/WADP-IS-Testing/2012/WADA_IST_2012_EN.pdf [9 April 2012] [17] V.A. Convertino. Blood volume response to physical activity and inactivity. Am. J. Med. Sci. 2007, 334, 72. [18] Y.O. Schumacher, F. Klodt, D. Nonis, T. Pottgiesser, M. Alsayrafi, P.C. Bourdon, S.C. Voss. The impact of long-haul air travel on variables of the athlete’s biological passport. Int. J. Lab. Hematol. 2012, 34, 641. [19] R.I. Holt. Detecting growth hormone abuse in athletes. Drug Test. Anal. 2009, 1, 426.
586 wileyonlinelibrary.com/journal/dta
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 582–586
