Protein and peptide hormones in anti-doping
When growth hormone (GH) is administered, it affects a number of hormones in the body, in particular insulin-like growth factor I (or IGF-I) produced mainly in the liver and amino-terminal propeptide of type III collagen (P-III-NP) produced during bone and soft tissue production and breakdown. Using IGF-I and P-III-NP measurements combined according to a mathematical formula, of these biomarkers, enables one to identify the administration of GH as distinct from natural production. Since both GH and the biomarkers decline with age, an age adjustment to the formula is used. A separate formula is required for males and females since sex also affects these biomarkers.
The World Anti-Doping Agency have approved the biomarker test based on blood serum samples (serum being the clear liquid obtained when blood is coagulated). There is a need also to be able to measure these biomarkers in whole blood, including dried blood samples, as well as blood plasma (cell free blood).
Sophisticated but very reliable methods (using liquid chromatography coupled mass spectrometry) have been validated and checked by two international collaborative studies for the measurement of IGF-I. A similar approach for P-III-NP is being developed by us.The administration of growth hormone (GH) is prohibited in sport as are some of the substances that affect natural GH release or effects. Growth hormone releasing factors (GHRF) is one such group of substances. They range in size from relatively small chemicals, which are quite simple to detect in doping control analysis, to a group of peptides (or small proteins) whose detection is far more complex. This research investigates whether the metabolites of the larger GHRFs might be better substances to prove GHRF administration.
The administration of insulin and the wide range of insulin analogues are prohibited in sport unless one has a therapeutic use exemption such as is granted to individuals with insulin-dependent diabetes. Their concentrations in blood and urine is small, which poses a particular challenge. Furthermore, they are readily “lost” from the sample. We have shown that at least some of this loss is because of degradation of the insulin and/or the insulin “sticking” to the test tubes used during sample handling.
Investigators
| Professor David Cowan | Principal Investigator |
| Lead Analyst Blood Doping | |
| Senior Analyst-Blood Doping | |
| Head of Science & Medicine, UK Anti-Doping (UKAD) | |
| University of Washington | |
| Chief Science Officer at US Anti-Doping Agency (USADA) | |
| Senior Research Scientist, SMRTL | |
| President/Director SMRTL, Salt Lake City | |
| Director UCLA Olympic Analytical Laboratory | |
|
Director, German Sport University Cologne |
|
| German Sport University Cologne | |
| University of Washington | |
| Emeritus Professor, University of Washington | |
| University of Southampton | |
|
Clinical Senior Lecturer, University Hospital Leicester |
|
| Consultant Clinical Toxicologist | |
|
Chief Science Officer, US Anti-Doping Agency (USADA) |
|
|
University of Southampton |
|
| Ivana Gavrilovic |
|
| Adam Tilcock |
|
| Scarlett Devey |
|
| Maggie (Malgorzata) Puchnarewicz |
|
| Dean Nyarko-Mensah |
|
PhD Students
| Siham Memdouh | PhD Student |
Aims
One:
- Quantification of Growth-Hormone-biomarkers (IGF-I and P-III-NP) in dried blood and blood plasma. Investigation to determine the relationship between the GH biomarkers in various blood matrices
- Determination of P-III-NP by LC-MS. To develop reference methods for quantifying P-III-NP in blood serum by LC-MS
Two:
- Identification of Growth-Hormone Releasing Factors (GHRF). Understand the metabolism of larger GHRFs (>3 kDa).
- Synthesis and characterisation of appropriate metabolites and develop sensitive analytical methods.
Three:
- Understanding the pre-analytical degradation of insulins. Insulins appear to be lost from samples by both degradation and adsorption. A better understanding of these losses is needed to improve insulin detection in sports samples. An understanding of the degradation is also needed to evaluate forensic cases where intoxication with an insulin is suspected.
Methods
Quantitative proteomics using LC-MS/MS
Trials Design
Double blind cross-over study design for GH administration (studies with University of Southampton)
Summary of Findings
Ongoing
Impact
These research projects support the objectives of anti-doping and will deliver methodologies suitable for the detection of a administered growth hormone, b GHRF in sport and c administered insulins in sport and other areas of forensic toxicology. Many of the methods will also be of value to the clinical community. The research underpinning this work was recorded as a Case Impact Study in the 2014 Research Excellence Framework: Catching the Drug Cheats: The Science Behind Anti-Doping for the London 2012 Olympic and Paralympic Games
https://ref2014impact.azurewebsites.net/casestudies2/refservice.svc/GetCaseStudyPDF/41268
Work that has informed Protein and peptide hormones in anti-doping includes:
- WADA Laboratory Guidelines Human Growth Hormone (hGH) Biomarkers Test
- https://www.wada-ama.org/en/resources/laboratories/guidelines-human-growth-hormone-hgh-biomarkers-test/
- https://www.wada-ama.org/en/questions-answers/human-growth-hormone-hgh-testing/
- https://www.wada-ama.org/sites/default/files/resources/files/tue_physician_guidelines_ghdchild_version2.2_july2020.pdf/
- Powrie, J. K., Bassett, E. E., Rosen, T., Jorgensen, J. O., Napoli, R., Sacca, L., Christiansen, J. S., Bengtsson, B. A., Sonksen, P. H., & Grp, G. H. P. S. Detection of growth hormone abuse in sport. Growth Horm. IGF Res. 2007; 17(3): 220-226.
Publications
- Moncrieffe D. Protein quantification relevant to antidoping. Bioanalysis. 2020 Jun;12(11):711-714. doi: 10.4155/bio-2020-0042. Epub 2020 Mar 25. PMID: 32209021.
- Moncrieffe D, Cox HD, Carletta S, Becker JO, Thomas A, Eichner D, Ahrens B, Thevis M, Bowers LD, Cowan DA, Hoofnagle AN. Inter-Laboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured Intact by Mass Spectrometry. Clin Chem. 2020 Apr 1;66(4):579-586. doi: 10.1093/clinchem/hvaa043. PMID: 32232452.
- Moncrieffe D, Parkin MC, Cowan DA. Peptide selection for the quantification of P-III-NP in human serum by mass spectrometry. Rapid Commun Mass Spectrom. 2018 Apr 15;32(7):535-542. doi: 10.1002/rcm.8066. PMID: 29361649.
- Holt RI, Böhning W, Guha N, Bartlett C, Cowan DA, Giraud S, Bassett EE, Sönksen PH, Böhning D. 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. Drug Test Anal. 2015 Sep;7(9):745-55. doi: 10.1002/dta.1772. Epub 2015 Jan 21. PMID: 25645199.
- Cox HD, Lopes F, Woldemariam GA, Becker JO, Parkin MC, Thomas A, Butch AW, Cowan DA, Thevis M, Bowers LD, Hoofnagle AN. Interlaboratory agreement of insulin-like growth factor 1 concentrations measured by mass spectrometry. Clin Chem. 2014 Mar;60(3):541-8. doi: 10.1373/clinchem.2013.208538. Epub 2013 Dec 9. PMID: 24323979.
- Cowan DA, Bartlett C. Laboratory issues in the implementation of the marker method. Growth Horm IGF Res. 2009 Aug;19(4):357-60. doi: 10.1016/j.ghir.2009.04.005. Epub 2009 Jul 1. PMID: 19574074.
- Kicman AT, Miell JP, Teale JD, Powrie J, Wood PJ, Laidler P, Milligan PJ, Cowan DA. Serum IGF-I and IGF binding proteins 2 and 3 as potential markers of doping with human GH. Clin Endocrinol (Oxf). 1997 Jul;47(1):43-50. doi: 10.1046/j.1365-2265.1997.2111036.x. PMID: 9302371.
Conferences
March 2021 [Ivana Gavrilovic] Manfred Donike Institute virtual Symposium. Oral presentation on Improving the detection of peptide hormones for anti-doping purposes.
October 2019 [David Cowan] 18th Annual United States Anti-Doping Agency Symposium on Anti-Doping Science. Invited speaker. Peptide quantification and future perspectives on anti-doping science.
April 2019 [Danielle Moncrieffe] Partnership for Clean Competition Conference on Anti-Doping, London. Invited speaker. GH Biomarkers IGF-I and P-III-NP: an update.
September 2018 [Danielle Moncrieffe] Reid Forum, Royal Society of Chemistry. Bio-Analytical Troubleshooting Workshop. Invited case study presentation. Improving the detection of insulin analysis in urine by LC-MS.
April 2018 [Danielle Moncrieffe- recipient of conference award] Manfred Donike Workshop, 36th Cologne Workshop on Dope Analysis. Peptide selection for the quantification of P-III-NP in human serum by mass spectrometry.
September 2017 [Danielle Moncrieffe- recipient of conference award] 38th British Mass Spectrometry Society Annual Meeting, Clinical and Forensic Analysis using Mass Spectrometry. Quantification of P-III-NP in humans: meeting the challenge.
September 2016 [David Cowan] Standards and Metrology for Anti-Doping Analysis. BIPM (International Bureau of Weights and Measures), Paris, France. Biomarker assay for hGH and the role of IGF-I measurements.
April 2015 [David Cowan] Partnership for Clean Competition Conference on Anti-Doping, New York, USA. Invited keynote speaker. Recent Breakthroughs in Detecting Human Growth Hormone Administration.
Work that has informed Protein and peptide hormones in anti-doping includes:
- WADA Laboratory Guidelines Human Growth Hormone (hGH) Biomarkers Test
- https://www.wada-ama.org/en/resources/laboratories/guidelines-human-growth-hormone-hgh-biomarkers-test/
- https://www.wada-ama.org/en/questions-answers/human-growth-hormone-hgh-testing/
- https://www.wada-ama.org/sites/default/files/resources/files/tue_physician_guidelines_ghdchild_version2.2_july2020.pdf/
- Powrie, J. K., Bassett, E. E., Rosen, T., Jorgensen, J. O., Napoli, R., Sacca, L., Christiansen, J. S., Bengtsson, B. A., Sonksen, P. H., & Grp, G. H. P. S. Detection of growth hormone abuse in sport. Growth Horm. IGF Res. 2007; 17(3): 220-226.
Publications
- Moncrieffe D. Protein quantification relevant to antidoping. Bioanalysis. 2020 Jun;12(11):711-714. doi: 10.4155/bio-2020-0042. Epub 2020 Mar 25. PMID: 32209021.
- Moncrieffe D, Cox HD, Carletta S, Becker JO, Thomas A, Eichner D, Ahrens B, Thevis M, Bowers LD, Cowan DA, Hoofnagle AN. Inter-Laboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured Intact by Mass Spectrometry. Clin Chem. 2020 Apr 1;66(4):579-586. doi: 10.1093/clinchem/hvaa043. PMID: 32232452.
- Moncrieffe D, Parkin MC, Cowan DA. Peptide selection for the quantification of P-III-NP in human serum by mass spectrometry. Rapid Commun Mass Spectrom. 2018 Apr 15;32(7):535-542. doi: 10.1002/rcm.8066. PMID: 29361649.
- Holt RI, Böhning W, Guha N, Bartlett C, Cowan DA, Giraud S, Bassett EE, Sönksen PH, Böhning D. 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. Drug Test Anal. 2015 Sep;7(9):745-55. doi: 10.1002/dta.1772. Epub 2015 Jan 21. PMID: 25645199.
- Cox HD, Lopes F, Woldemariam GA, Becker JO, Parkin MC, Thomas A, Butch AW, Cowan DA, Thevis M, Bowers LD, Hoofnagle AN. Interlaboratory agreement of insulin-like growth factor 1 concentrations measured by mass spectrometry. Clin Chem. 2014 Mar;60(3):541-8. doi: 10.1373/clinchem.2013.208538. Epub 2013 Dec 9. PMID: 24323979.
- Cowan DA, Bartlett C. Laboratory issues in the implementation of the marker method. Growth Horm IGF Res. 2009 Aug;19(4):357-60. doi: 10.1016/j.ghir.2009.04.005. Epub 2009 Jul 1. PMID: 19574074.
- Kicman AT, Miell JP, Teale JD, Powrie J, Wood PJ, Laidler P, Milligan PJ, Cowan DA. Serum IGF-I and IGF binding proteins 2 and 3 as potential markers of doping with human GH. Clin Endocrinol (Oxf). 1997 Jul;47(1):43-50. doi: 10.1046/j.1365-2265.1997.2111036.x. PMID: 9302371.
Conferences
March 2021 [Ivana Gavrilovic] Manfred Donike Institute virtual Symposium. Oral presentation on Improving the detection of peptide hormones for anti-doping purposes.
October 2019 [David Cowan] 18th Annual United States Anti-Doping Agency Symposium on Anti-Doping Science. Invited speaker. Peptide quantification and future perspectives on anti-doping science.
April 2019 [Danielle Moncrieffe] Partnership for Clean Competition Conference on Anti-Doping, London. Invited speaker. GH Biomarkers IGF-I and P-III-NP: an update.
September 2018 [Danielle Moncrieffe] Reid Forum, Royal Society of Chemistry. Bio-Analytical Troubleshooting Workshop. Invited case study presentation. Improving the detection of insulin analysis in urine by LC-MS.
April 2018 [Danielle Moncrieffe- recipient of conference award] Manfred Donike Workshop, 36th Cologne Workshop on Dope Analysis. Peptide selection for the quantification of P-III-NP in human serum by mass spectrometry.
September 2017 [Danielle Moncrieffe- recipient of conference award] 38th British Mass Spectrometry Society Annual Meeting, Clinical and Forensic Analysis using Mass Spectrometry. Quantification of P-III-NP in humans: meeting the challenge.
September 2016 [David Cowan] Standards and Metrology for Anti-Doping Analysis. BIPM (International Bureau of Weights and Measures), Paris, France. Biomarker assay for hGH and the role of IGF-I measurements.
April 2015 [David Cowan] Partnership for Clean Competition Conference on Anti-Doping, New York, USA. Invited keynote speaker. Recent Breakthroughs in Detecting Human Growth Hormone Administration.
Principal Investigators
Danielle Moncrieffe
Research Associate, Drug Control Centre
Vincenzo Abbate
Reader in Bioanalysis
Affiliations
Funding
Funding Body: Partnership for Clean Competition
Amount: £235,000
Period: September 2019 - September 2022